Novel PD1-targeted IL-2 immunocytokine and VITOKINE fusions

Abstract

The present disclosure provides novel PD1 Ab-IL-2 immunocytokine and VitoKine compositions aimed at targeting potent or bioactivatable IL-2 directly to tumor-infiltrating lymphocytes to reduce systemic mechanism-based toxicity and lead to broader therapeutic utility for IL-2 in cancer treatment. With PD1 Ab-IL-2 VitoKines, IL-2 remains inactive until activated locally by proteases upregulated in diseased tissues, preventing pathway overactivation and reducing undesirable "on-target" and "off-tissue" toxicity, as well as significantly reducing potential antigen or target sinks, thereby resulting in prolonged in vivo half-life and improved biodistribution, bioavailability, and therapeutic efficacy. Additionally, PD1 antibodies with the ability to block PD1 and reverse T cell anergy or exhaustion may further cooperate with IL-2 anti-cancer immune responses.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 404,608, filed September 8, 2022, which is incorporated herein by reference in its entirety.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (SeqListing-CUGENE PD1 AB-IL-2.xml; size: 216 kb; created on: September 5, 2023) are incorporated herein by reference in their entirety.

[0003] Technical Field While cancer has traditionally been treated with chemotherapy, radiation, targeted therapy, and surgery, the recent emergence of immunotherapy, a fifth pillar of cancer treatment, has transformed the fight against cancer. The benchmark for immunotherapeutic drugs has been established with the development of T cell checkpoint (CTLA-4 and PD1 / PD-L1) inhibitors. These treatments have been demonstrated to effectively expand and reactivate the pool of tumor-specific T cells, leading to objective response rates of up to 50% in patients with certain cancers. [Background technology]

[0004] Interleukin-2 (IL-2) was the first growth factor described for T cells. IL-2's ability to expand lymphocyte populations in vivo and increase the effector functions of these cells confers antitumor effects to IL-2 and led to the approval of high-dose recombinant IL-2 for certain metastatic cancers. Although durable responses have been demonstrated in approximately 10% of patients, IL-2 cancer immunotherapy is associated with multiple problems, including severe toxicity caused by the induction of vascular leak syndrome (VLS), tumor tolerance caused by the induction of activation-induced cell death (AICD), and immunosuppression caused by the activation of Treg cells.

[0005] Several approaches have been taken to overcome the inherent challenges of IL-2 immunotherapy. One such approach to combat systemic toxicity involves localizing cytokine activity to cancer cells and their surrounding tissues through tumor-targeted IL-2 immunocytokines, constructed by fusing IL-2 to antibodies specific for tumor-associated antigens. However, this strategy lacks the ability to specifically target effector T cells within the tumor microenvironment (TME), which are relevant to anti-cancer immunity. This gap in intratumoral T cell targeting can be filled by fusing IL-2 to anti-programmed cell death protein 1 (PD1) antibodies. PD1 (also known as CD279) is highly expressed on tumor-infiltrating lymphocytes (TILs), and PD1 antibody-IL-2 immunocytokines allow IL-2 to be directly targeted to TILs. It exhibits increased avidity for intratumoral CD8+ T cells, but not for Treg cells or peripheral CD4+ and CD8+ T cells. This strategy therefore further improves IL-2 anti-cancer immunity while reducing systemic toxicity.

[0006] In addition to directly targeting IL-2 to TILs to improve IL-2 anti-cancer immunity, PD1 antibodies capable of blocking PD1 and reversing T cell anergy or exhaustion can cooperate with IL-2 to further boost the anti-cancer immune response. Therefore, it is desirable to construct a PD1 Ab-IL-2 immunocytokine with a PD1 antibody that has excellent target binding and PD1 blocking capabilities. Among the various globally marketed PD1 blocking antibodies that have fundamentally transformed the field of cancer immunotherapy, pembrolizumab (Keytruda®; Merck Sharp & Dohme Corp.) has received significant attention due to its high efficacy and approval for treating various cancer types. Although pembrolizumab exhibits excellent target binding and blocking capabilities, it has several sequence disadvantages, including a relatively low degree of affinity, which may raise immunogenicity concerns, and high hydrophobicity, which tends to increase its aggregation tendency. Therefore, it is preferable to optimize pembrolizumab to mitigate these sequence disadvantages while fully maintaining its biological activity. The resulting optimized sequence is expected to improve the developability of PD1 Ab-IL-2 fusion proteins.

[0007] Importantly, fusion of PD1 Ab with a fully active IL-2 moiety can abolish the intended antibody-mediated targeting, causing the fusion protein to localize to IL-2 receptor-expressing cells in the periphery instead of TILs in the tumor. Therefore, to improve target specificity and selectivity, one approach is to prepare fusions using an IL-2 moiety with attenuated IL-2Rβγ activity to establish a stoichiometric balance between the cytokine and antibody components. Additionally, reducing cytokine potency can potentially alleviate pathway overactivation as well as alleviate antigen sink and target-mediated deposition.

[0008] Another, related but more sophisticated strategy for improving target specificity and selectivity is to apply the VitoKine platform disclosed by the present inventors in International Publication Nos. 2019246392 and 2021119516. In the VitoKine construct, the activity of the IL-2 moiety remains inactive or minimal until locally activated by proteases upregulated in or around the tumor. By doing so, binding of the IL-2 moiety to its receptor in the periphery or on the cell surface of non-diseased cells can be significantly limited. This can help prevent pathway overactivation and reduce undesirable "on-target" and "off-tissue" toxicity, and VitoKine's improved safety profile may allow for human dose levels within the effective range of PD1 antibodies. Additionally, the inactivity of the IL-2 moiety prior to protease activation significantly reduces potential antigens or target sinks and therefore results in a prolonged in vivo half-life and improved biodistribution and bioavailability at the intended site of treatment. Summary of the Invention

[0009] Disclosure of the Invention In one embodiment, the present invention provides a novel PD1-targeted bioactivatable IL-2 immunocytokine (referred to herein as PD1 Ab-IL-2 VitoKine) designed to target bioactivatable IL-2 directly to tumor-infiltrating lymphocytes. The activity of the IL-2 moiety remains largely inactive or minimal until locally activated by proteases upregulated in the tumor, which limits the binding of the IL-2 moiety to its receptors in the periphery or on the cell surface of non-diseased or normal cells. This can help prevent pathway overactivation, reduce undesired "on-target" and "off-tissue" toxicity, and minimize unwanted target sinks.

[0010] In another aspect, the present invention provides a novel PD1-targeted IL-2 immunocytokine that aims to target the activity-modulated IL-2 domain directly to tumor-infiltrating lymphocytes. The attenuated IL-2 activity is expected to facilitate the establishment of a stoichiometric balance between the cytokine and antibody arms, help alleviate pathway overactivation, and mitigate antigen sink and target-mediated deposition.

[0011] The strategy specifically targets effector T cells within the tumor microenvironment (TME) that are relevant to anti-cancer immunity. By implementing this strategy, the ability of IL-2 to expand lymphocyte populations and increase their effector function is combined with the function of PD1-blocking antibodies in reversing T cell anergy or exhaustion. This approach, particularly when attenuated or bioactivatable IL-2 is used, reduces systemic mechanism-based toxicity, leading to broader therapeutic utility of IL-2 for cancer treatment, as well as improving biodistribution and bioavailability at the intended site of treatment.

[0012] In various embodiments, the PD1-targeting bioactivatable IL-2 immunocytokine is referred to herein as a PD1 Ab-IL-2 VitoKine. In various embodiments, the VitoKine platform disclosed by the inventors in WO2019246392 and WO2021119516 is defined by the construct depicted in Figure 1 and one of the proposed activation methods depicted in Figure 2. In various embodiments, the PD1 Ab-IL-2 VitoKine of the present invention is more particularly defined by the construct shown in Figure 3A. Referring to Figure 3A, the PD1 Ab-IL-2 VitoKine of the present invention comprises a PD1-blocking antibody, a monovalent IL-2 domain (active partial domain) whose N-terminus is fused to the C-terminus of the heterodimeric Fc chain of the PD1 antibody via an L1 linker, and whose C-terminus is fused to the N-terminus of the IL-2Rα sushi domain (hidden partial domain) via an L2 linker.

[0013] In various embodiments, the variable domains of the PD1-blocking antibodies of the invention were optimized from the variable domain of pembrolizumab by introducing germline sequence substitutions into CDR residues, introducing germline sequence substitutions into framework somatic mutations, and / or adopting the most prevalent and better-behaved VH3 human germline family sequences as acceptor frameworks. In various embodiments, the PD1-blocking antibodies have high affinity for the human PD1 protein set forth in SEQ ID NO: 1, function to inhibit PD1 with equal or comparable potency to pembrolizumab, exhibit a higher sequence similarity score to its closest human germline sequence than pembrolizumab, thereby indicating an improved degree of humanity, and are predicted to have lower hydrophobicity than pembrolizumab, which in turn reduces the tendency to aggregate.

[0014] In various embodiments, a PD1 blocking antibody comprises a light chain variable region having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region having the sequence set forth in SEQ ID NO:7. In various embodiments, a PD1 blocking antibody comprises a light chain variable region having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region having the sequence set forth in SEQ ID NO:9. In various embodiments, a PD1 blocking antibody comprises a light chain variable region having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region having the sequence set forth in SEQ ID NO:11. In various embodiments, a PD1 blocking antibody comprises a light chain variable region having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region having the sequence set forth in SEQ ID NO:13. In various embodiments, a PD1 blocking antibody comprises a light chain variable region having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region having the sequence set forth in SEQ ID NO:18.

[0015] In various embodiments, the PD1-targeted IL-2 immunocytokine is defined by the construct depicted in Figure 3B. In various embodiments, the potency-modulating IL-2 of the PD1-targeted IL-2 immunocytokine is an IL-2 variant (or mutant) comprising a sequence derived from the sequence of the mature human IL-2 polypeptide set forth in SEQ ID NO: 116 (also referred to herein as huIL-12 or IL-2 wild-type (w / t)), which contains one or more amino acid substitutions, deletions, or insertions. In various embodiments, the amino acid changes are one or more amino acid substitutions at positions 19, 65, 125, or 126 of SEQ ID NO: 116. In various embodiments, the amino acid change is a substitution of L with D or H or N or P or Q or R or S or Y at position 19 of the mature human IL-2 sequence, a substitution of P with G or E or H or R or A or K or N or Q at position 65, a substitution of C with I at position 125, a substitution of Q with A or D or E or F or G or H or I or K or L or M or N or P or R or S or T or V or W or Y at position 126, or any combination of these substitutions. In various embodiments, the IL-2 variant has reduced / eliminated binding to IL-2Rα compared to a native IL-2 polypeptide. In various embodiments, the IL-2 variant has decreased binding activity for the IL-2Rβγ receptor compared to a native IL-2 polypeptide. In various embodiments, the IL-2 variant has both reduced / eliminated binding to IL-2Rα and modulated binding activity for the IL-2Rβγ receptor compared to a native IL-2 polypeptide. In various embodiments, the IL-2 variant is selected from the group of sequences set forth in SEQ ID NOs: 117-180.

[0016] In various embodiments, the active portion of the PD1 Ab-IL-2 VitoKine is an IL-2 domain comprising the sequence of the mature human IL-2 polypeptide set forth in SEQ ID NO: 116. In various embodiments, the IL-2 domain is an IL-2 variant (or mutant) comprising a sequence derived from the sequence of the mature human IL-2 polypeptide set forth in SEQ ID NO: 116, comprising one or more amino acid substitutions, deletions, or insertions. In various embodiments, the amino acid changes are one or more amino acid substitutions at positions 19, 65, 125, or 126 of SEQ ID NO: 116. In various embodiments, the amino acid change is a substitution of L with D or H or N or P or Q or R or S or Y at position 19 of the mature human IL-2 sequence, a substitution of P with G or E or H or R or A or K or N or Q at position 65, a substitution of C with I at position 125, a substitution of Q with A or D or E or F or G or H or I or K or L or M or N or P or R or S or T or V or W or Y at position 126, or any combination of these substitutions. In various embodiments, the VitoKine construct comprises an IL-2 moiety designed to have reduced / eliminated binding to IL-2Rα. In various embodiments, the IL-2 variant has reduced binding activity for IL-2Rβγ compared to the native IL-2 polypeptide. In various embodiments, the IL-2 variant has both reduced / eliminated binding to IL-2Rα and altered binding activity for IL-2Rβγ compared to the native IL-2 polypeptide. In various embodiments, the IL-2 variant in the VitoKine construct can tune the basal endogenous IL-2 VitoKine activity to achieve optimal anti-tumor efficacy while minimizing unwanted systemic toxicity due to an expanded therapeutic window. In various embodiments, the IL-2 domain is selected from the group of sequences set forth in SEQ ID NOs: 117-180.

[0017] In various embodiments, the cryptic partial domain is a cognate receptor / binding partner or any binding partner identified for IL-2. In various embodiments, the cryptic partial domain is the IL-2Rα extracellular domain having the sequence set forth in SEQ ID NO: 181, or a functional fragment thereof. In various embodiments, the IL-2Rα extracellular domain, or a functional fragment thereof, is the IL-2Rα sushi domain having the sequence set forth in SEQ ID NO: 182. In various embodiments, the cryptic partial domain is a variant (mutant) of the IL-2Rα sushi domain. In various embodiments, the amino acid changes are one or more amino acid substitutions at positions 36, 38, 42, or 43 of SEQ ID NO: 182. In various embodiments, the amino acid changes are an R for A at position 36, a K for E at position 38, an L for G at position 42, or a Y for A at position 43. In various embodiments, the variant (mutant) of the IL-2Rα sushi domain is designed to facilitate dissociation and diffusion after proteolytic cleavage. In various embodiments, the variant (mutant) of the IL-2Rα Sushi domain is selected from the group of sequences set forth in SEQ ID NOs: 183-185.

[0018] In various embodiments, the L1 linker and the L2 linker of the PD1 Ab-IL-2 VitoKine construct are both protease-cleavable peptide linkers. In various embodiments, L1 of the PD1 Ab-IL-2 VitoKine construct is a protease-cleavable peptide linker, and L2 is a non-cleavable peptide linker. In various embodiments, L1 of the PD1 Ab-IL-2 VitoKine construct is a non-cleavable peptide linker, and L2 is a protease-cleavable peptide linker. In various embodiments, the L1 linker and the L2 linker of the PD1 Ab-IL-2 VitoKine construct are both protease-non-cleavable peptide linkers. In various embodiments, the non-cleavable linker is rich in G / S content (e.g., at least about 60%, 70%, 80%, or 90% or more of the amino acids in the linker are G or S). Each peptide linker sequence can be selected independently. In various embodiments, the protease-cleavable linker is selected from the group of sequences set forth in SEQ ID NOs: 54-77. In various embodiments, the protease-cleavable linker can have an additional peptide spacer of variable length on the N-terminus of the cleavable linker or on the C-terminus of the cleavable linker or on both ends of the cleavable linker to improve accessibility for enzymatic cleavage. In various embodiments, the protease-cleavable linker having a peptide spacer of variable length on either the N-terminus or the C-terminus of the cleavable linker or on both ends is selected from the group of sequences set forth in SEQ ID NOs: 78-94. In various embodiments, the non-cleavable linker is selected from the group of sequences set forth in SEQ ID NOs: 95-115. In various embodiments, the linker is either flexible or rigid and of various lengths.

[0019] In various embodiments, the IL-2 domain (D2) and the IL-2Rα domain (D3) of the VitoKine construct are placed C-terminal to the PD1 Ab domain (D1) as depicted in Figure 1A. In various embodiments, the D2 and D3 domains of the VitoKine construct are placed N-terminal to the D1 domain as depicted in Figure 1B.

[0020] In various embodiments, the PD1 blocking Ab, IL-2 domain, and IL-2Rα domain of the PD1 Ab-IL-2 VitoKine construct can be monomers or dimers or a combination of dimers and monomers, for example, the PD1 blocking Ab is a dimer and the IL-2 domain and IL-2Rα domain are monomers.

[0021] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need of cancer or cancer metastasis treatment.In one embodiment, the subject is a human subject.In various embodiments, the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, or rhabdomyosarcoma or any cancer.

[0022] In another aspect, the present disclosure provides a method of treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention in combination with a second treatment selected from the group consisting of cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, stem cell transplantation, cell therapy including chimeric antigen receptor (CAR)-T, CAR-NK, induced pluripotent stem cell (iPS)-derived CAR-T or iPS-derived CAR-NK, and a vaccine, e.g., Bacillus Calmette-Guerin (BCG). In various embodiments, the combination therapy includes treatments using depleting antibodies against specific tumor antigens; treatments using antibody-drug conjugates; treatments using agonist, antagonist, or blocking antibodies against costimulatory or co-inhibitory molecules (immune checkpoints), such as CTLA-4, PD-L1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec-7, Siglec-8, Siglec-9, Siglec-15, and VISTA; treatments using bispecific T cell engaging antibodies (BiTE®), such as blinatumomab; treatments involving administration of biological response modifiers, such as IL-12, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ; treatments using therapeutic vaccines, such as sipuleucel-T; The combination treatment may include administering to the subject a therapeutically effective amount of immunotherapy, including, but not limited to, treatment using a cellular vaccine or a tumor antigen peptide vaccine; treatment using CAR-T cells; treatment using CAR-NK cells; treatment using tumor infiltrating lymphocytes (TIL); treatment using adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic); treatment using TALL-104 cells; and treatment using immunostimulants, such as Toll-like receptor (TLR) agonists CpG and imiquimod; and treatment using a vaccine, such as BCG; the combination treatment provides increased effector cell killing of tumor cells, i.e., synergism exists between the VitoKine construct and the immunotherapy when administered in combination.

[0023] In another aspect, the present disclosure provides a use of a pharmaceutical composition of the present invention for the preparation of a medicament for the treatment of cancer.

[0024] In another aspect, the present disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding the pharmaceutical composition of the present disclosure. In another aspect, the present disclosure provides a vector comprising the nucleic acid described herein. In various embodiments, the vector is an expression vector. In another aspect, the present disclosure provides an isolated cell comprising the nucleic acid of the present disclosure. In various embodiments, the cell is a host cell comprising an expression vector of the present disclosure. In another aspect, a method for producing a VitoKine construct by culturing a host cell under conditions that promote expression of a protein or polypeptide is provided.

[0025] In another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated pharmaceutical composition of the present invention in admixture with a pharmaceutically acceptable carrier. [Brief explanation of the drawings]

[0026] [Figure 1] Figure 1 depicts representative VitoKine construct formats. Figure 1A depicts a VitoKine construct in which D2 (active moiety domain) and D3 (hidden moiety domain) are placed C-terminal to D1 (targeting domain). Figure 1B depicts a VitoKine construct in which the D2 and D3 domains are placed N-terminal to the D1 domain. [Figure 2]Figure 2 depicts a proposed activation mechanism for the PD1 Ab-IL-2 VitoKine construct of the present invention. An exemplary VitoKine construct contains two protease-cleavable linkers; protease 1 activation resulting from cleavage of the L1 linker results in active form 1; protease 2 activation resulting from cleavage of the L2 linker results in active form 2; and activation by both proteases resulting from cleavage of the L1 and L2 linkers results in active form 3. After protease cleavage, the hidden moiety domain (D3) is released and diffuses away from the active moiety domain (D2). When the L1 linker is the only protease-cleavable linker, active form 1 is the only activated format. Similarly, when the L2 linker is the only protease-cleavable linker, active form 2 is the single activated format. [Figure 3] Figure 3A depicts a representative PD1 Ab-IL-2 VitoKine construct of the present invention. Monomeric IL-2 or IL-2 variant as the active partial domain (D2) is fused at its N-terminus to the C-terminus of the PD1 antibody heterodimeric Fc (D1) using an L1 linker; the C-terminus of the IL-2 moiety is fused to the N-terminus of IL-2Rα or IL-2Rα variant as the hidden partial domain (D3) using an L2 linker. Figure 3B depicts a representative PD1 Ab-IL-2 immunocytokine that also serves as a non-VitoKine immunocytokine counterpart. [Figure 4] Figure 4 depicts a comparison of PD1-blocking activity between a reference antibody (P-0734) and a pembrolizumab (PBL) biosimilar in a luciferase reporter assay. Figures 4A and 4B depict the dose-dependent increase in luminescence signal and fold induction, respectively. P-0734 and the PBL biosimilar share identical variable domains and have IgG1 and IgG4 isotypes, respectively. [Figure 5]Figure 5 depicts (A) ELISA binding and (B-C) PD1 blocking activity of PD1 blocking antibodies P-1148, P-1150, P-1151, and P-1153 compared to a reference antibody (P-0734) when tested in a luciferase reporter assay. Figure 5B and Figure 5C depict the dose-dependent increase in luminescence signal and fold induction, respectively. [Figure 6] Figure 6 depicts the PD1-blocking activity of PD1-blocking antibodies P-1127, P-1129, and P-1174 compared to a reference antibody (P-0734), which were tested in a luciferase reporter assay and show a dose-dependent increase in luminescence signal. [Figure 7] Figure 7 depicts the PD1-blocking activity of PD1-blocking antibodies, P-1175 and P-1181, compared to a reference antibody (P-0734), when tested in a luciferase reporter assay. Figures 7A and 7B depict the dose-dependent increase in luminescence signal and fold induction, respectively. [Figure 8] Figure 8 depicts the PD1-blocking activity of PD1-blocking antibodies P-1175, P-1176, P-1177, and P-1178 compared to a reference antibody (P-0734) when tested in a luciferase reporter assay. Figures 8A and 8B depict the dose-dependent increase in luminescence signal and fold induction, respectively. [Figure 9] Figure 9 depicts the PD1-blocking activity of PD1-blocking antibodies P-1198, P-1199, and P-1201 compared to a reference antibody (P-0734) when tested in a luciferase reporter assay. Figures 9A and 9B depict the dose-dependent increase in luminescence signal and fold induction, respectively. A non-targeting germline antibody was included as a negative control. [Figure 10]Figure 10 depicts the PD1-blocking activity of PD1-blocking antibodies P-1194, P-1201, and P-1238 compared to a reference antibody (P-0734) when tested in a luciferase reporter assay. Figures 10A and 10B depict the dose-dependent increase in luminescence signal and fold induction, respectively. [Figure 11] Figure 11 depicts the binding of PD1-blocking antibodies P-1174, P-1193, P-1198, P-1199, and P-1201 to PD1+ HEK293 cells compared to a reference antibody (P-0734). Figures 11A and 11C depict the dose-dependent increase in the percentage of positive cells, and Figures 11B and 11D depict the dose-dependent increase in mean fluorescence intensity (MFI). [Figure 12] Figure 12 depicts the ELISA binding of IL-2RαSushi variants, P-0751, P-0752, and P-0753, to IL-2. P-0757 contains wild-type IL-2RαSushi and was included for comparison. [Figure 13] Figure 13 depicts the activity evaluation of Fc IL-2 VitoKine with either wild-type IL-2RαSushi (P-0701) or IL-2RαSushi variants (P-0754, P-0755, and P-0756) as the D3 domain. P-0704, an IL-2 P65R variant Fc fusion protein, was included as a fully active IL-2 control. Activity was assessed by analyzing the induction of Ki67 expression on A) CD8+ T cells and B) NK cells of human PBMCs using flow cytometry. [Figure 14] Figure 14 depicts ELISA binding of IL-2 variants to IL-2Rα. Each IL-2 variant harbors a different amino acid substitution at position P65 (see Table 18 for details regarding molecular information). P-0531 and P-0689 serve as wild-type IL-2 controls in bivalent and monovalent forms, respectively. [Figure 15]Figure 15 depicts the efficacy of IL-2 P65 variants in stimulating STAT5 phosphorylation in CD4+ Treg cells (see Table 18 for details on molecular information). P-0531 and P-0689 serve as wild-type IL-2 controls in bivalent and monovalent forms, respectively. Similarly, Benchmark (D) and Benchmark (E) are bivalent and monovalent forms of the IL-2 F42A / Y45A / L72G variant, respectively. [Figure 16] Figure 16 depicts the activity of IL-2 P65 variants towards IL-2Rβγ by analyzing A) ELISA binding to recombinantly expressed IL-2 receptor subunit β and γ complexes, and B) induction of Ki67 expression on CD8+ T cells in fresh human PBMCs using flow cytometry. See Table 18 for details on molecular information of IL-2 variants. P-0531 and P-0689 serve as wild-type IL-2 controls in bivalent and monovalent forms, respectively. [Figure 17] Figure 17 depicts the activity assessment of various surrogate murine PD1 Ab-IL-2 VitoKines, P-0800, P-0830, P-0831, and P-0802, compared to P-0782, a non-VitoKine immunocytokine counterpart. This was performed by analyzing the induction of Ki67 expression on A) CD8+ T cells and B) NK cells of human PBMCs. The four IL-2 antibody VitoKines differ only in the binding strength of their IL-2 partial domain to IL-2Rα. [Figure 18] Figure 18 depicts the activity evaluation of IL-2 variants P-0731, P-0759, and P-0761, which harbor mutations that disrupt interaction with IL-2Rβ. The evaluation was based on their effect on inducing Ki67 expression on A) CD8+ T cells and B) NK cells of human PBMCs. All these IL-2 variants also have the P65R mutation, which eliminates binding to IL-2Rα. P-0704 serves as a fully active IL-2 control. [Figure 19]Figure 19 depicts the activity evaluation of IL-2 variants harboring mutations that interfere with binding to γc. The evaluation was based on their effect on inducing Ki67 expression on CD8+ T cells (A, C, & E) and NK cells (B, D, F) of human PBMCs. All these IL-2 variants also have the P65R mutation, which eliminates IL-2 Ra binding. P-0704 serves as a fully active IL-2 control. [Figure 20] Figure 20 depicts the activity evaluation of the IL-2 variant P-1247, harboring mutations targeting both IL-2Rβ and γc, compared to P-1158, which contains only γc-interfering mutations. Evaluation was performed by analyzing the effect on inducing Ki67 expression on A) CD8+ T cells and B) NK cells of human PBMCs. P-0704 serves as an IL-2 full agonist control. [Figure 21] Figure 21 depicts the PD1 blocking activity of PD1 blocking antibodies P-1174, P-1238, and P-1271 compared to the respective PD1 Ab-IL-2 VitoKine, P-1197, P-1239, and P-1272, in a luciferase reporter assay. Figures 21A and 21B depict the dose-dependent increase in luminescence signal and fold induction, respectively. [Figure 22] Figure 22 depicts the evaluation of the endogenous basal IL-2 activity of PD1 Ab-IL-2 VitoKines P-0872, P-1197, and P-1272 compared to their corresponding non-VitoKine immunocytokine counterparts, P-0879 and P-1271. Activity evaluation was based on analyzing the induction of the proliferation marker Ki67 on CD8+ T cells (A&C) and NK cells (B&D) of human PBMCs. The three VitoKines differ only in D1 domain composition, i.e., they contain distinct PD1-blocking antibodies. P-1174, a constituent PD1 antibody of P-1197, was included as a negative control for the assay. [Figure 23]Figure 23 depicts protease cleavage and activation of PD1 Ab-IL-2 VitoKine. The figure includes A) a reducing SDS-PAGE gel showing both the intact and active forms of P-1272, along with demonstration of dose-dependent induction of Ki67 expression on CD8+ T cells for B) VitoKine P-1272 versus its non-VitoKine counterpart P-1273, C) VitoKine P-0831 versus its non-VitoKine counterpart P-0838, and D) VitoKine P-1345 versus its non-VitoKine counterpart P-0838. [Figure 25] Figure 25 depicts the dose- and time-dependent effects of a single dose of P-0831, a murine PD1 Ab-IL-2 VitoKine, on the expansion of A) CD8+ T cells, B) Granzyme B+ CD8+ T cells, C) NK cells, and D) Granzyme B+ NK cells in the peripheral blood of C57B / L6 mice. P-0838, its non-VitoKine immunocytokine counterpart, was included for comparison. Blood was collected on days 0, 3, 5, 7, and 10 for lymphocyte phenotyping by FACS analysis. Data are presented as mean ± SEM. [Figure 26] Figure 26 depicts the effect of a single dose of P-0831, a murine PD1 Ab-IL-2 VitoKine, on (A) the dose-dependent increase in serum levels of the inflammatory marker IFNγ, and (B) the change in body weight between different dosing levels in naive C57BL / 6 mice. P-0838, its non-VitoKine immunocytokine counterpart, was included for comparison. Additionally, vehicle (PBS) and its constituent murine PD1 antibody, P-0722, were used as negative controls. [Figure 27]Figure 27 depicts the antitumor effect of P-0831, a murine PD1 Ab-IL-2 VitoKine, in an established MC38 mouse colon cancer model after two doses administered once every 10 days (Q10D). MC38 tumor growth curves in individual mice are presented for A) 3 mg / kg P-0831, B) 6 mg / kg P-0831, C) 9 mg / kg P-0831, and D) 1 mg / kg P-0838, its non-VitoKine immunocytokine counterpart. For comparison, the mean tumor volume over time ± standard error of the mean (SEM) for the vehicle group is plotted as a dotted line. The mean tumor volume over time ± SEM for each treatment group is shown in Figure 27E, and the change in body weight over time for each treatment group is shown in Figure 27F. [Figure 28] Figure 28 depicts immunohistochemistry (IHC) analysis of the effects of P-0831, a murine PD1 Ab-IL-2 VitoKine, on tumor tissue isolated 5 days after treatment at a dose of 6 mg / kg. Tissue sections were fixed in 10% formalin, paraffin-embedded, processed, and stained with antibodies by HistoWiz to assess immune cells in the tumor tissue. For comparison, P-0722, its component murine PD1 antibody, at a dose of 6 mg / kg, and P-0838, its non-VitoKine immunocytokine counterpart, at a dose of 1 mg / kg were included. [Figure 29] Figure 29 depicts the ex vivo activity in human PBMCs and in vivo antitumor efficacy in mice bearing established CT26 mouse tumors of P-0831 compared to its non-cleavable VitoKine counterpart, P-0877. In vitro assessment measures induction of the proliferation marker Ki67 on A) CD8+ T cells and B) NK cells in fresh human PBMCs. In vivo analysis includes changes in C) mean tumor volume ± SEM and D) body weight over time for each treatment group in the CT26 mouse model after two doses of 10 mg / kg Q12D. P-0879 was used in the human PBMC assay as a fully active IL-2 immunocytokine control. P-0722, the component murine PD1 antibody of P-0831, dosed at 10 mg / kg, was included as a control in the tumor model. [Figure 30] Figure 30 depicts the in vivo anti-tumor efficacy of P-0831 compared to its non-targeting VitoKine counterpart, P-0871, and its component murine PD1 antibody, P-0722, in mice bearing established CT26 mouse tumors. Mean tumor volume ± SEM was plotted as a function of time for each treatment group in the CT26 mouse model after two doses of 10 mg / kg Q12D. [Figure 31] FIG. 31 depicts the activity assessment of murine PD1 Ab-IL-2 immunocytokines, P-0782, P-0783, and P-0786, by analyzing their effects on A) induction of Ki67 expression on human CD8+ T cells, B) induction of Ki67 expression on human NK cells, and C) proliferation of murine CTLL-2 cells. [Figure 32] Figure 32 depicts the dose-dependent and temporal pharmacodynamic effects of several murine PD1 Ab-IL-2 immunocytokines, including A) peripheral CD8+ T cell expansion, B) peripheral NK cell expansion, and C) changes in body weight after a single dose of 2 mg / kg in C57B / L6 mice. Blood was collected on days 0, 3, 5, 7, and 10 for lymphocyte phenotyping by FACS analysis. Data are presented as mean ± SEM. [Figure 33] Figure 33 depicts the antitumor efficacy of several murine PD1 Ab-IL-2 immunocytokines in an established MC38 mouse colon cancer model after two treatments with Q12D at 0.5 mg / kg. The mean tumor volume ± SEM over time for each treatment group is shown in Figure 33A, and individual tumor volumes in mice 26 days after the first dose are shown in Figure 33B. [Figure 34]Figure 34 depicts the antitumor efficacy of two murine PD1 Ab-IL-2 immunocytokines, P-0783 and P-0786, in an established MC38 mouse colon cancer model after two doses of Q10D at 0.3 mg / kg. The mean tumor volume ± SEM over time for each treatment group is shown in Figure 34A. Individual growth curves of subcutaneous MC38 tumors in mice are shown for B) P-0783 and C) P-0786. [Figure 35] Figure 35 depicts the antitumor efficacy of P-0786, a murine PD1 Ab-IL-2 immunocytokine, in an established MC38 mouse colon cancer model after two treatments with Q10D at 1 mg / kg. This includes the change in A) mean tumor volume ± SEM and B) body weight over time for each treatment group. The component murine PD1 antibody, P-0722, dosed at 9 mg / kg, was included for comparison. [Figure 36] Figure 36 depicts the dose-dependent antitumor efficacy of P-0786, a murine PD1 Ab-IL-2 immunocytokine, in an established MC38 mouse colon cancer model after two treatments with Q14D at varying dose levels. The mean tumor volume ± SEM over time for each treatment group is shown in Figure 36A. Individual tumor growth curves are shown for B) 0.03 mg / kg, C) 0.1 mg / kg, C) 0.3 mg / kg, and E) 1 mg / kg doses. The mean tumor volume ± SEM over time for the vehicle group (represented as a dotted line) is included for comparison. [Figure 37] Figure 37 depicts the absence of tumor recurrence after rechallenge with MC38 colon cancer cells in tumor-free mice treated with P-0786, in contrast to the successful regrowth of the same type of tumor in age-matched naive mice as controls. [Figure 38] Figure 38 depicts the efficacy of P-0786 as a single agent in inhibiting tumor growth in two additional subcutaneous syngeneic tumor models. These models include A) the CT26 mouse colon carcinoma tumor model and B) the B16F10 mouse melanoma tumor model. DETAILED DESCRIPTION OF THE INVENTION

[0027] In one embodiment, the present disclosure provides a PD1 Ab-IL-2 VitoKine construct comprising three domains: an optimized PD1 blocking antibody as the TIL targeting moiety, an IL-2 variant as the active moiety, and an IL-2 Ra sushi variant as the masking moiety. Importantly, the IL-2 Ra sushi variant domain has the ability to mask or attenuate the functional activity of the IL-2 domain until it is activated at the site of intended treatment.

[0028] PD1-blocking antibodies guide VitoKine to TILs in the tumor microenvironment and locally restrict VitoKine activation, improving the therapeutic index. In various embodiments, PD1-blocking antibodies were optimized through modifications in the variable domain of pembrolizumab. In various embodiments, the modifications involved germline sequence substitution of CDR residues, germline sequence substitution of framework residues, and adoption of VH3 human germline family sequences as the acceptor framework. In various embodiments, these modifications were implemented individually or in combination to develop optimized PD1-blocking antibodies. In various embodiments, these optimized PD1-blocking antibodies exhibit high binding affinity to PD1, function to inhibit PD1 with equal or comparable efficacy to pembrolizumab, have a higher sequence similarity score to their closest human germline sequence, resulting in an improved degree of affinity compared to pembrolizumab, and have lower hydrophobicity, leading to a reduced tendency to aggregate. In various embodiments, PD1 Ab-IL-2 VitoKine constructs based on these optimized PD1 blocking antibodies have enhanced developability.

[0029] In various embodiments, the IL-2 domain is the active moiety but remains inactive until locally activated by proteases upregulated in diseased tissues; this limits the binding of the active moiety to receptors in the periphery or on the cell surface of non-diseased cells or tissues, preventing pathway overactivation and reducing undesirable "on-target" and "off-tissue" toxicity. VitoKine's improved safety profile may allow for human dose levels within the effective range of PD1 antibodies. Additionally, the inactivity of the VitoKine active moiety prior to protease activation significantly reduces potential antigen sinks and therefore prolongs in vivo half-life, resulting in improved biodistribution, bioavailability, and efficacy at the intended site of treatment.

[0030] In various embodiments, the incorporation of a potency-attenuated IL-2 variant as an active moiety domain (such an IL-2 variant being achieved by disrupting the IL-2Rβγ interaction) can further fine-tune the intrinsic basal activity and activity of VitoKine after activation. In various embodiments, such VitoKine with a potency-attenuated IL-2 variant as an active moiety domain may additionally expand its therapeutic index.

[0031] In various embodiments, the unique and non-signaling α-subunit of the IL-2 receptor is used as a cryptic moiety via a protease-cleavable linker to reversibly occlude cytokine activity. It may be preferable for the cryptic α-subunit to dissociate after protease cleavage of the linker. Consequently, amino acid modifications of the α-receptor to modulate binding affinity for IL-2 may be beneficial.

[0032] In various embodiments, in the PD1 Ab-IL-2 VitoKine construct, the three domains are linked using two linkers of variable length and rigidity and are optionally coupled to protease-cleavable sequences. These protease-cleavable sequences are peptide substrates for specific protease subtypes with elevated or deregulated expression in disease sites, thereby allowing the functional IL-2 domain to appear or be released at the site of disease. The length and composition of the linker were fine-tuned to ensure optimal hiding of the IL-2 domain from access to its receptor, thereby minimizing systemic engagement. Meanwhile, the stability of the VitoKine construct in the blood circulation was maintained while allowing efficient cleavage upon encounter with specific proteases at the intended site of treatment.

[0033] In another aspect, the present disclosure provides novel PD1-targeting IL-2 immunocytokines that target tumor-infiltrating lymphocytes directly through the active-modulated IL-2 domain. In various embodiments, the PD1-blocking antibodies are optimized through modifications in the variable domain of pembrolizumab. In various embodiments, the optimized PD1-blocking antibody-based targeted IL-2 immunocytokines are predicted to have enhanced development potential.

[0034] In various embodiments, an activity-modulated IL-2 domain (monomer) is fused to the C-terminus of a heterodimeric PD1 antibody heavy chain. In various embodiments, the IL-2 domain in the PD1-targeted IL-2 immunocytokine is IL-2Rβγ-selective and attenuated. In various embodiments, attenuated IL-2 potency facilitates the establishment of a stoichiometric balance between the cytokine and antibody components, helps reduce pathway overactivation, and alleviates antigen sink and target-mediated deposition. In various embodiments, the use of an attenuated IL-2 variant (such a variant has impaired interaction with γc) in a PD1-targeted IL-2 immunocytokine may provide an additional benefit in alleviating antigen sink and, in turn, may result in an extended in vivo half-life, possibly due to the influence of the γc receptor in the signaling cascade leading to cell proliferation.

[0035] definition Unless otherwise defined herein, scientific and technical terms used in the context of the present invention have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms include plurals, and plural terms include the singular. Generally, the technical terms used in the context of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, and these techniques, are commonly used and well known in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in the various general and more specific references referenced and discussed throughout this specification. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), incorporated herein by reference. Enzymatic reactions and purification techniques are carried out according to manufacturer's specifications as commonly accomplished in the art or as described herein. The technical terms used in connection with, and laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those commonly used and well known in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.

[0036] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. In various embodiments, a "peptide," "polypeptide," and "protein" is a chain of amino acids whose alpha carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (the amino terminus) thus has a free amino group, and the terminal amino acid at the other end of the chain (the carboxy terminus) has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated as N-terminus) refers to the free α-amino group on the amino acid at the amino terminus of a peptide or the α-amino group (the amino group participating in a peptide bond) of an amino acid at any other position within the peptide. Similarly, the term "carboxy terminus" (abbreviated as C-terminus) refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other position within the peptide. Peptides also include essentially any polyamino acid, including, but not limited to, peptidomimetics, e.g., those in which amino acids are joined by ether bonds rather than amide bonds.

[0037] Polypeptides of the present disclosure include polypeptides that have been modified in any way and for any reason, such as, for example, (1) to reduce susceptibility to proteolysis, (2) to reduce susceptibility to oxidation, (3) to alter binding affinity for forming protein complexes, (4) to alter binding affinity, and (5) to impart or modify other physicochemical or functional properties.

[0038] As used herein, an amino acid "substitution" refers to the replacement of one amino acid in a polypeptide at a specific position in a parent polypeptide sequence with a different amino acid. Amino acid substitutions can be generated using genetic or chemical methods well known in the art. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in a naturally occurring sequence (e.g., in a portion of a polypeptide outside the domain that forms intermolecular contacts). A "conservative amino acid substitution" refers to the replacement of an amino acid with a functionally similar amino acid in a polypeptide. The following six groups each contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Serine (S), and Threonine (T) 2) Aspartic acid (D) and glutamic acid (E) 3) Asparagine (N) and Glutamine (Q) 4) Arginine (R) and Lysine (K) 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V) 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)

[0039] "Non-conservative amino acid substitutions" refer to the replacement of a member of one of these classes with a member from another class. In making such changes, according to various embodiments, the hydropathic index of amino acids may be considered. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0040] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (see, e.g., Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certain amino acids can be substituted with other amino acids having a similar hydropathic index or score and still retain similar biological activity. When making changes based on hydropathic index, various embodiments include substitution of amino acids whose hydropathic index is within ±2, in various embodiments within ±1, and in various embodiments within ±0.5.

[0041] It is also understood in the art that substitutions of like amino acids can be usefully made on the basis of hydrophilicity, particularly when the resulting biologically functional protein or peptide is intended for use in immunological embodiments, as disclosed herein. In various embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[0042] The following hydrophilicity values ​​have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5), and tryptophan (-3.4). When making changes based on similar hydrophilicity values, various embodiments include substitutions of amino acids whose hydrophilicity values ​​are within ±2, various embodiments include substitutions within ±1, and various embodiments include substitutions within ±0.5.

[0043] Exemplary amino acid substitutions are listed in Table 1. TIFF2025531804000001.tif178170

[0044] Those skilled in the art can use well-known techniques to determine suitable variants of the polypeptides described herein. In various embodiments, those skilled in the art can identify suitable sections of the molecule that can be changed without destroying activity by targeting regions that are not believed to be important for activity. In other embodiments, those skilled in the art can identify residues and portions of the molecule that are conserved between similar polypeptides. In further embodiments, even sections that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure.

[0045] Additionally, one skilled in the art can review structure-function studies to identify residues in similar polypeptides that are important for activity or structure. In light of such comparisons, one skilled in the art can predict the importance of amino acid residues in a polypeptide that correspond to amino acid residues in the similar polypeptide that are important for activity or structure. One skilled in the art can select substitutions of chemically similar amino acids for such predicted important amino acid residues.

[0046] Those skilled in the art can also analyze the three-dimensional structure and the amino acid sequence relative to that structure in similar polypeptides. Taking such information into account, those skilled in the art can predict the alignment of amino acid residues of a polypeptide relative to the three-dimensional structure of the polypeptide. In various embodiments, those skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the polypeptide, because such residues may be involved in important interactions with other molecules. Furthermore, those skilled in the art may generate test variants containing single amino acid substitutions at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if it is discovered that a change to a specific amino acid residue results in destroyed, undesirably reduced, or unsuitable activity, the variant with such a change can be avoided. In other words, based on the information gathered from such routine experiments, those skilled in the art can easily determine amino acids for which further substitutions should be avoided, either alone or in combination with other mutations.

[0047] The terms "polypeptide fragment" and "truncated polypeptide," as used herein, refer to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion compared to the corresponding full-length protein. In various embodiments, the fragment can be, for example, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids in length. In various embodiments, fragments can also be, for example, up to 1000, up to 900, up to 800, up to 700, up to 600, up to 500, up to 450, up to 400, up to 350, up to 300, up to 250, up to 200, up to 150, up to 100, up to 50, up to 25, up to 10, or up to 5 amino acids in length. A fragment can further comprise one or more additional amino acids at either or both of its termini, for example, a sequence of amino acids from a different naturally occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

[0048] The terms "polypeptide variant," "hybrid polypeptide," and "polypeptide mutant," as used herein, refer to a polypeptide comprising an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted compared to another polypeptide sequence. In various embodiments, the number of inserted, deleted, or substituted amino acid residues can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids in length. Hybrids of the present disclosure include fusion proteins.

[0049] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, e.g., conjugated to another chemical moiety, e.g., polyethylene glycol, albumin (e.g., human serum albumin), etc., phosphorylated, and glycosylated.

[0050] The term "sequence identity %" is used interchangeably herein with the term "identity %" and refers to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same as 80% sequence identity determined by a defined algorithm, meaning that a given sequence is at least 80% identical to another sequence. In various embodiments, the identity % is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence identity to a given sequence. In various embodiments, the percent identity is within the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0051] The term "sequence identity %" is used interchangeably herein with the term "homology %" and refers to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same as 80% sequence identity determined by a defined algorithm, and thus, homologs of a given sequence have a sequence identity greater than 80% over a certain length of the given sequence. In various embodiments, the identity % is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence identity to the given sequence. In various embodiments, the percent homology is within the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0052] Exemplary computer programs that can be used to determine the identity between two sequences include, but are not limited to, the BLAST series of programs published on the NCBI website on the Internet, such as BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN.Also see Altschul et al., J. Mol. Biol. 215:403-10, 1990 (particularly referring to the published default settings, i.e., parameters w=4, t=17) and Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997.Sequence searches are typically performed using the BLASTP program, where a given amino acid sequence is compared with the amino acid sequences in GenBank Protein Sequences and other public databases.The BLASTX program is preferred for searching nucleic acid sequences translated in all reading frames against the amino acid sequences in GenBank Protein Sequences and other public databases. Both BLASTP and BLASTX are run using default parameters of an open gap penalty of 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix.

[0053] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability in the comparison of the test nucleic acid with the reference nucleic acid is, for example, less than about 0.1, less than about 0.01, or less than about 0.001.

[0054] The term "alteration" (modification), as used herein, refers to any manipulation of the peptide backbone (eg, amino acid sequence) or post-translational modification of a polypeptide (eg, glycosylation).

[0055] The term "knobs-into-hole modification" as used herein refers to a modification in the interface between two immunoglobulin heavy chains in the CH3 domain.In one embodiment, the "knobs-into-hole modification" comprises the amino acid substitution T366W and optionally the amino acid substitution S354C in one of the antibody heavy chains, and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain.Knobs-into-hole technology is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng 9,617-621 (1996) and Carter, J Immunol Meth 248,7-15 (2001).

[0056] The term "bioactivatable drug" or "VitoKine," as used herein, refers to a compound that is a drug precursor, which, after administration to a subject, releases the drug in vivo through some chemical or physiological process such that the bioactivatable drug is converted into a product that is active on the target tissue. A bioactivatable drug is any compound that undergoes bioactivation before exhibiting its pharmacological effect. A bioactivatable drug can therefore be viewed as a drug containing specialized, non-toxic protecting groups that are used in a transient manner to modify or eliminate undesirable properties in the parent molecule.

[0057] The term "immunoconjugate" or "fusion protein," as used herein, refers to a molecule comprising an antibody or its antigen-binding fragment directly or indirectly conjugated (or linked) to an effector molecule. The effector molecule can be a detectable label, immunotoxin, cytokine, chemokine, therapeutic agent, or chemotherapeutic agent. The antibody or its antigen-binding fragment may be conjugated to the effector molecule via a peptide linker. The immunoconjugate and / or fusion protein retains the immunoreactivity of the antibody or antigen-binding fragment; for example, the antibody or antigen-binding fragment has approximately the same, or only slightly reduced, ability to bind to an antigen after conjugation as before conjugation. As used herein, an immunoconjugate may also be referred to as an antibody-drug conjugate (ADC). Because immunoconjugates and / or fusion proteins are originally prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they may also be referred to as "chimeric molecules."

[0058] A "linker" refers to a molecule that joins two other molecules covalently or through ionic, van der Waals, or hydrogen bonds, e.g., a nucleic acid molecule that hybridizes at its 5'-end to one complementary sequence and at its 3'-end to another complementary sequence, thereby joining two non-complementary sequences. A "cleavable linker" refers to a linker that can be degraded, digested, or otherwise cleaved to separate the two components connected by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, and lipases. Cleavable linkers may also be cleaved by environmental cues, such as changes in temperature, pH, salt concentration, and the like.

[0059] The term "peptide linker," as used herein, refers to a peptide comprising one or more amino acids, typically about 1 to 30 amino acids. Peptide linkers are known in the art or are described herein. Suitable non-immunogenic linker peptides include, for example, (G4S) n , (SG4) n or G4 (SG4) n It includes a peptide linker. "n" is generally a number from 1 to 10, typically from 2 to 4.

[0060] A "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in animals. A pharmaceutical composition contains a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. A "pharmacologically effective amount" refers to an amount of an agent effective to produce the intended pharmacological result. A "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, vehicles, buffers, and excipients, such as phosphate-buffered saline solution, a 5% aqueous solution of dextrose, and emulsions, such as oil / water or water / oil emulsions, as well as various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st Ed. 2005, Mack Publishing Co., Easton. A "pharmaceutically acceptable salt" is a salt that can be formulated into a compound for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0061] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and may be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, remission or palliation of the disease state, and improvement in remission or prognosis. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder, or condition. Furthermore, references herein to "treatment" include references to curative, palliative, and preventative treatment.

[0062] The term "effective amount" or "therapeutically effective amount" as used herein refers to the amount of a compound or composition that is sufficient to treat a specified disorder, condition or disease, for example, to ameliorate, alleviate, reduce, and / or delay one or more of its symptoms.With respect to cancer or other unwanted cell proliferation, an effective amount includes: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow down, and preferably stop the cancer cell invasion into peripheral organs to some extent; (iv) inhibit (i.e., slow down and preferably stop to some extent) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumor; and / or (vii) alleviate to some extent one or more of the symptoms associated with cancer.An effective amount can be administered in one or more administrations.

[0063] The phrases "administering" or "causing to be administered" refer to the act of managing and / or allowing the administration of an agent / compound of interest to a patient by a medical professional (e.g., a physician) or someone managing the medical care of a patient. Causing to be administered can involve determining a diagnosis and / or an appropriate treatment regimen, and / or prescribing a particular agent / compound for the patient. Such prescribing can include, for example, issuing a prescription, annotating a medical record, and the like. When administering is described herein, "causing to be administered" is also contemplated.

[0064] The terms "patient," "individual," and "subject" may be used interchangeably and may refer to a mammal, preferably a human or non-human primate, but may also refer to domestic mammals (e.g., canines or felines), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., equines, bovines, porcines, ovines). In various embodiments, a patient may be a human (e.g., an adult male, adult female, adolescent male, adolescent female, boy, girl) under the care of a physician or other healthcare professional in a hospital, psychiatric care facility, outpatient setting, or other clinical setting. In various embodiments, a patient may be an immunocompromised patient or a patient with a weakened immune system, including, but not limited to, patients with primary immunodeficiency, AIDS; cancer and transplant patients receiving certain immunosuppressive drugs; and patients with genetic diseases affecting the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient has an immunogenic cancer, including, but not limited to, bladder cancer, lung cancer, melanoma, and other cancers that have been reported to have high rates of mutations (Lawrence et al., Nature, 499(7457):214-218, 2013).

[0065] The term "immunotherapy" refers to treatments using depleting antibodies against specific tumor antigens; treatments using antibody-drug conjugates; treatments using costimulatory or co-inhibitory molecules (immune checkpoints), such as CTLA-4, PD1, PDL-1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, SIRPα, CD47, GITR, ICOS, CD27, Siglec 7, Siglec 8, Siglec 9, Siglec treatments using agonist, antagonist, or blocking antibodies against 15, VISTA, CD276, CD272, TIM-3, and B7-H4; treatments using bispecific T cell engaging antibodies (BiTE®), such as blinatumomab; treatments involving administration of biological response modifiers, such as IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, IL-22, GM-CSF, IFN-α, IFN-β, IFN-γ, TGF-β antagonists, or TGF-β traps; treatments using therapeutic vaccines, such as sipuleucel-T; treatments using therapeutic viruses, including, but not limited to, oncolytic viruses, such as T-vec; dendritic cell vaccines, or tumor antigen peptides or treatment using neoantigen vaccines; treatment using NK cells; treatment using chimeric antigen receptor (CAR)-T cells; treatment using CAR-NK cells; treatment using DCs or T cells; treatment using iPS-induced NK cells; treatment using iPS-induced T cells; treatment using vaccines, such as Bacillus Calmette-Guerin (BCG); treatment using tumor-infiltrating lymphocytes (TILs); treatment using adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR-T cells); treatment using TALL-104 cells; and treatment using immunostimulants, such as Toll-like receptor (TLR) agonists CpG, TLR7, TLR8, TLR9, and imiquimod.

[0066] "Resistant or refractory cancer" refers to tumor cells or cancers that do not respond to prior anti-cancer treatments, including, for example, chemotherapy, surgery, radiation therapy, stem cell transplantation, and immunotherapy. Tumor cells can be resistant or refractory at the beginning of treatment, or they may become resistant or refractory during treatment. Refractory tumor cells include tumors that do not respond at the start of treatment or that initially respond to treatment for a short period but then become unresponsive. Refractory tumor cells also include tumors that respond to treatment with an anti-cancer therapy but do not respond to subsequent rounds of treatment. For purposes of the present invention, refractory tumor cells also encompass tumors that appear to be inhibited by treatment with an anti-cancer therapy but recur up to five years, sometimes up to ten years, or even longer after treatment has ceased. Anti-cancer treatments can use chemotherapy alone, radiation alone, targeted therapy alone, surgery alone, or a combination thereof. For ease of description and not limitation, it is understood that refractory tumor cells can be interchangeable with resistant tumors.

[0067] The term "neoantigen" refers to cell surface antigens to which the immune system has not previously been exposed, e.g., that are selectively expressed by cancer cells or overexpressed in cancer cells relative to most normal cells, particularly those that result from alteration of host antigens by radiation, chemotherapy, viral infection, neoplastic transformation / mutation, drug metabolism, etc.

[0068] The term "antibody," as used herein, is used in the broadest sense and encompasses a variety of antibody structures (IgG1, 2, 3, or 4, IgM, IgA, IgE), including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific or bifunctional antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0069] The term "antibody fragment," as used herein, refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab'), diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies.

[0070] The term "Fab fragment," as used herein, refers to an immunoglobulin fragment containing the VL domain and the constant domain of the light chain (CL), and the VH domain and the first constant domain of the heavy chain (CH1).

[0071] The term "variable region" or "variable domain," as used herein, refers to the domain of an immunoglobulin or antibody heavy or light chain that is generally responsible for binding the immunoglobulin or antibody to an antigen. The variable domains of the heavy and light chains of immunoglobulins or antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three complementarity-determining regions (CDR).

[0072] The term "complementarity-determining region" or "CDR" refers to the antigen-contacting residues ("antigen contacts"). Antibodies generally contain six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). The CDRs are located at amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), 89-97 (CDR-L3), 31-35b (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity-determining residues (SDRs).

[0073] A "single-chain antibody" is an Fv molecule in which the heavy and light chain variable regions are connected by a flexible linker to form a single polypeptide chain that forms the antigen-binding region. Single-chain antibodies are discussed in detail in WO 88 / 01649, U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference.

[0074] A "human immunoglobulin," as used herein, is an immunoglobulin having an amino acid sequence that corresponds to that of an immunoglobulin produced by a human or human cell, or derived from a non-human source that utilizes the human immunoglobulin repertoire or other human immunoglobulin-coding sequences. This definition of a human immunoglobulin specifically excludes humanized immunoglobulins that contain non-human antigen-binding residues.

[0075] The term "humanized antibody" as used herein refers to an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDR. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of amino acid substitutions taken from the donor framework, and such substitutions are referred to herein as back mutations. A humanized or other monoclonal antibody may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions.

[0076] The term "Fc domain" or "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term includes native-sequence Fc regions and variant Fc regions. The IgG Fc region includes the IgG CH2 and IgG CH3 domains. The CH3 region herein may be a native-sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "protuberance" ("knob") introduced in one chain and a corresponding "cavity" ("hole") introduced in the other chain; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical immunoglobulin heavy chains as described herein. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system.

[0077] The term "effector function," as used herein, refers to a biological activity attributed to the Fc region of an immunoglobulin, which varies with immunoglobulin isotype. Examples of immunoglobulin effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0078] As used herein, "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an immunoglobulin to bind to a specific antigen can be measured either through enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology.

[0079] The term "affinity" or "binding affinity" as used herein refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (KD), which is the ratio of the dissociation and association rate constants (koff and kon, respectively). A particular method for measuring affinity is SPR.

[0080] The term "immunogenicity," as used herein, refers to the ability of an antibody or antigen-binding fragment to elicit an immune response (humoral or cellular) when administered to a recipient, including, for example, a human anti-mouse antibody (HAMA) response. A HAMA response is initiated when T cells from a subject mount an immune response to an administered antibody. The T cells then recruit B cells to produce specific "anti-antibody" antibodies.

[0081] The term "immune cell," as used herein, refers to any cell of the hematopoietic lineage that is involved in regulating the immune response to an antigen (e.g., an autoantigen). In various embodiments, the immune cell is, for example, a T cell, a B cell, a dendritic cell, a monocyte, a natural killer cell, a macrophage, a Langerhans cell, or a Kupffer cell.

[0082] The term "reduced binding," as used herein, refers to a decrease in affinity for the respective interaction, e.g., as measured by SPR. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

[0083] The term "polymer," as used herein, generally includes, but is not limited to, homopolymers; copolymers, such as block, graft, random, and alternating copolymers; and terpolymers; and blends and modifications thereof. Furthermore, unless otherwise specifically limited, the term "polymer" includes all possible geometric configurations of the material. These configurations include, but are not limited to, isotactic, syndiotactic, and random symmetries.

[0084] "Polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs. Nucleic acid analogs include nucleic acid analogs containing non-naturally occurring bases, nucleotides containing bases that are linked to other nucleotides in linkages other than naturally occurring phosphodiester bonds, or that are attached through linkages other than phosphodiester bonds. Thus, nucleotide analogs include, without limitation, for example, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to short polynucleotides, generally of about 50 nucleotides or less. Where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it is understood that this also includes RNA sequences in which "U" replaces "T" (i.e., A, U, G, C).

[0085] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of nucleotides to a nascent RNA transcript is referred to as the transcription direction. A DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand having the same sequence as the mRNA transcribed from that DNA and which are located 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; and sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."

[0086] "Complementary" refers to the topological compatibility or matching of the interacting surfaces of two polynucleotides. Therefore, the two molecules can be described as complementary, and further, the characteristics of the contacting surfaces are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions.

[0087] A "vector" is a polynucleotide that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), in which additional DNA segments can be introduced into the viral genome. Certain vectors have the ability to autonomously replicate in host cells into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and thereby are replicated along with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0088] A "regulatory sequence" is a nucleic acid that affects the expression (e.g., level, timing, or location of expression) of a nucleic acid to which it is operably linked. A regulatory sequence can exert its effect, for example, directly on the regulated nucleic acid or through the action of one or more other molecules (e.g., a polypeptide that binds to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., and Baron et al., 1995, Nucleic Acids Res. 23:3605-06. A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the nucleotide sequence.

[0089] A "host cell" is a cell that can be used to express a polynucleotide of the present disclosure. A host cell can be a prokaryote, such as E. coli, or a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. Typically, a host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, and the nucleic acid can then be expressed in the host cell. The phrase "recombinant host cell" can be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains a nucleic acid but does not express the nucleic acid at a desired level until a regulatory sequence is introduced into the host cell so that the nucleic acid is operably linked to the nucleic acid. It is understood that the term host cell refers not only to the specific subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due, for example, to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term as used herein.

[0090] The term "isolated molecule" (wherein the molecule is, for example, a polypeptide or polynucleotide) refers to a molecule that, by reason of its origin or source of derivation, (1) is not associated with naturally associated components that accompany it in its native state; (2) is substantially free from other molecules from the same species; (3) is expressed by cells from a different species; or (4) is not naturally occurring. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell from which it naturally originates is "isolated" from its naturally associated components. A molecule may also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule may be assayed by numerous means well known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, greater resolution may be provided by using HPLC or other means well known in the art for purification.

[0091] A protein or polypeptide is "substantially pure," "substantially homogeneous," or "substantially purified" when at least about 60%-75% of a sample represents a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein typically comprises about 50%, 60%, 70%, 80%, or 90% w / w of a protein sample, more usually about 95% w / w, and is preferably greater than 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by staining the gel with stains well known in the art to visualize a single polypeptide band. For certain purposes, greater resolution may be provided by using HPLC or other means well known in the art for purification.

[0092] The term "label" or "labeled," as used herein, refers to the incorporation of another molecule into an antibody. In one embodiment, the label is the incorporation of a detectable marker, such as a radiolabeled amino acid, or the attachment of a biotinyl moiety to the polypeptide that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In another embodiment, the label or marker can be therapeutic, such as a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include the following: radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents, e.g., gadolinium chelates, toxins, e.g., pertussis toxin, taxanes, Examples of suitable anti-cancer drugs include, but are not limited to, cyclohexyl 1, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. In various embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0093] The term "heterologous," as used herein, refers to a composition or condition that is not native or not found in nature, e.g., that may be achieved by replacing an existing natural composition or condition with a composition or condition derived from another source. Similarly, expression of a protein in an organism other than the organism in which the protein is naturally expressed constitutes a heterologous expression system and heterologous protein.

[0094] It is understood that aspects and embodiments of the disclosure described herein include aspects and embodiments that "consist of" and / or "consist essentially of."

[0095] Reference herein to "about" a value or parameter includes (and describes) variations directed to the value or parameter itself. For example, a reference to "about X" includes a description of "X."

[0096] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. It is understood that embodiments and variations of the disclosure described herein include "consisting of" and / or "consisting essentially of" embodiments and variations.

[0097] PD1 blocking antibodies In one embodiment, the PD1-blocking antibody guides the IL-2 portion of VitoKine to TILs in the tumor microenvironment (TME) and locally restricts VitoKine activation, improving the therapeutic index. In another embodiment, the PD1-blocking antibody guides the IL-2 portion of the immunocytokine to TILs in the TME. In various embodiments, the PD1-blocking antibody was optimized through modifications in the variable domain of pembrolizumab. In various embodiments, the modifications involved germline sequence substitution of CDR residues, germline sequence substitution of framework residues, and adoption of the most prevalent and better-performing VH3 human germline family sequence as the acceptor framework. In various embodiments, these modifications were implemented individually or in combination to develop an optimized PD1-blocking antibody. In various embodiments, these optimized PD1 blocking antibodies are predicted to exhibit high binding affinity to PD1, function to inhibit PD1 with equal or comparable potency to pembrolizumab, have a higher sequence similarity score to their closest human germline sequence, resulting in an improved degree of humanity compared to pembrolizumab, and have lower hydrophobicity, leading to a reduced tendency to aggregate compared to pembrolizumab. In various embodiments, PD1 Ab-IL-2 VitoKine constructs and PD1-targeted IL-2 immunocytokines based on these optimized PD1 blocking antibodies are predicted to have enhanced developability. In various embodiments, the PD1 antibody comprises a light chain variable region having a sequence selected from the group of sequences set forth in SEQ ID NOS: 3-5 and a heavy chain variable region having a sequence selected from the group of sequences set forth in SEQ ID NOS: 7-18. In various embodiments, the PD1 antibody comprises a light chain sequence set forth in SEQ ID NOS: 44 and a heavy chain having a sequence selected from the group of sequences set forth in SEQ ID NOS: 45-49.

[0098] IL-2 domain Interleukin-2 (IL-2), a classical Th1 cytokine, is produced by T cells after activation through the T cell antigen receptor and the costimulatory molecule CD28. IL-2 regulation occurs through activation of signaling pathways and transcription factors that act on the IL-2 promoter to generate new gene transcription, but also involves modulation of IL-2 mRNA stability. IL-2 binds to a multichain receptor containing a highly regulated α chain and β and γ chains that mediate signal transduction through the Jak-STAT pathway. IL-2 transmits activation, proliferation, and differentiation signals to T cells, B cells, and NK cells. IL-2 is also important in mediating activation-induced cell death of T cells, a function that provides an essential mechanism for terminating immune responses. A commercially available non-glycosylated human recombinant IL-2 product, aldesleukin (available as PROLEUKIN® brand of des-alanyl-1, serine-125 human interleukin-2 from Prometheus Laboratories Inc., San Diego, Calif.) has been approved for administration to patients with metastatic renal cell carcinoma and metastatic melanoma. IL-2 has also been suggested for administration in patients with or infected with hepatitis C virus (HCV), human immunodeficiency virus (HIV), acute myeloid leukemia, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, juvenile rheumatoid arthritis, atopic dermatitis, breast cancer, and bladder cancer. Unfortunately, the short half-life and severe toxicity limit optimal dosing of IL-2.

[0099] As used herein, the terms "native IL-2" and "native interleukin-2" in the context of a protein or polypeptide refer to any naturally occurring mammalian interleukin-2 amino acid sequence, including immature or precursor and mature forms. Non-limiting examples of GenBank accession numbers for the amino acid sequences of various species of native mammalian interleukin-2 include NP_032392.1 (mouse (Mus musculus), immature form), NP_001040595.1 (rhesus monkey (macaca mulatta), immature form), NP_000577.2 (human, precursor form), CAA01199.1 (human, immature form), and AAD48509.1 (human, immature form). In various embodiments of the present invention, native IL-2 is the immature or precursor form of naturally occurring mammalian IL-2. In other embodiments, native IL-2 is the mature form of naturally occurring mammalian IL-2. In various embodiments, native IL-2 is the precursor form of naturally occurring human IL-2. In various embodiments, native IL-2 is the mature form of naturally occurring human IL-2. In various embodiments, the IL-2 in the VitoKine and immunocytokine constructs of the present invention is derived from the amino acid sequence of the mature human IL-2 sequence set forth in SEQ ID NO: 116: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 116)

[0100] In various embodiments, the IL-2 domain is an IL-2 variant (or mutant) comprising a sequence derived from the sequence of the mature human IL-2 polypeptide set forth in SEQ ID NO: 116. In various embodiments, the IL-2 variant comprises a single C125I amino acid substitution, which universally enhances the developability of the protein while fully preserving biological activity. In various embodiments, the IL-2 variant comprising the single C125I mutation has the amino acid sequence set forth in SEQ ID NO: 117.

[0101] In various embodiments, the sequence of an IL-2 variant has at least one amino acid change, e.g., a substitution or deletion, compared to the native IL-2 sequence, resulting in IL-2 agonist or antagonist activity. An IL-2 agonist is exemplified by equivalent or increased biological activity compared to wild-type IL-2. An IL-2 antagonist is exemplified by decreased biological activity compared to wild-type IL-2 or the ability to inhibit IL-2-mediated responses. In various embodiments, the IL-2 variant has an amino acid sequence derived from SEQ ID NO: 117 with altered binding to IL-2Rα. In various embodiments, the IL-2 variant with altered binding to IL-2Rα comprises the amino acid sequences set forth in SEQ ID NOs: 118-125. In various embodiments, the IL-2 variant has an amino acid sequence derived from SEQ ID NO: 117 with reduced / eliminated binding to IL-2Rα to selectively activate and expand effector T cells (Teff) to treat cancer. In various embodiments, the IL-2 variant with reduced / eliminated binding to IL-2Rα comprises an amino acid sequence set forth in SEQ ID NOs: 118-122. In various embodiments, the IL-2 variant has an amino acid sequence derived from SEQ ID NO: 117 with reduced binding to IL-2Rβ and / or γc. In various embodiments, the IL-2 variant with reduced binding to IL-2Rβ and / or γc comprises an amino acid sequence set forth in SEQ ID NOs: 126-150. In various embodiments, the IL-2 variant has an amino acid sequence derived from SEQ ID NO: 117 with reduced / eliminated binding to IL-2Rα and reduced binding to IL-2Rβ and / or γc. In various embodiments, the IL-2 variant with reduced / eliminated binding to IL-2Rα and reduced binding to IL-2Rβ and / or γc comprises an amino acid sequence set forth in SEQ ID NOs: 151-180. As will be appreciated by one of skill in the art, all of the mutations can be optionally and independently combined in any way to achieve optimal affinity and activity modulation.

[0102] IL-2Rα domain (hidden domain in PD1 Ab-IL-2 VitoKine) The IL-2 receptor (IL-2R) is a heterotrimeric protein expressed on the surface of certain immune cells, such as lymphocytes, that binds and responds to IL-2. IL-2R has three subunits: α (CD25), β (CD122), and γ (CD132 or common gamma chain (γc)); a chain shared with five other cytokine receptors: IL-4R, IL-7R, IL-9R, IL-15R, and IL-21R. The alpha chain of the human receptor (also known as Tac antigen or p55) is encoded by the gene IL-2RA on chromosome 10p14-15. The gene for the human beta chain of the receptor (IL-2RB, CD122) is located on chromosome 22q11.2-12, and the gene for the human gamma chain (IL-2RG) is located on chromosome Xq13. Assembly of all three receptor subunits is important for signal transduction to B and T cells. IL-2R is found (either transiently or permanently) on the cell surface of almost all hematopoietic cells, including lymphoid T, B, and NK cells, as well as myeloid lineage cells such as macrophages, monocytes, and neutrophils. Signals are transduced into cells via Janus kinases Jak1 and Jak3. Phosphorylation of the intracytosolic portion of the receptor's β chain allows the homodimerization of STAT-3 and STAT-5 factors. STAT-3 and STAT-5 homodimers exhibit increased affinity for the nucleus, where they bind to specific DNA elements that enhance the transcription of IL-2-dependent genes.

[0103] As used herein, the terms "native IL-2Rα" and "native interleukin-2 receptor alpha" in the context of a protein or polypeptide refer to any naturally occurring mammalian interleukin-2 receptor alpha ("IL-2Rα") amino acid sequence, including immature or precursor and mature forms and naturally occurring isoforms. Non-limiting examples of GenBank accession numbers for the amino acid sequences of various native mammalian IL-2Rα include NP_032393.3 (mouse), CAK26553.1 (human), and NP_000408.1 (human). In various embodiments, the IL-2Rα domain is derived from the amino acid sequence of the human IL-2Rα sequence set forth in SEQ ID NO: 181: MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI (SEQ ID NO: 181)

[0104] In various embodiments, a masking partial domain (D3) is used to reversibly mask the activity of the IL-2 domain in the PD1 Ab-IL-2 VitoKine construct. In various embodiments, the masking partial domain is the IL-2Rα extracellular domain or a functional fragment thereof. In various preferred embodiments, the masking partial domain is an IL-2Rα Sushi domain comprising the amino acid sequence of the mature human IL-2Rα polypeptide set forth in SEQ ID NO: 182. In various preferred embodiments, the masking partial domain is a variant of the IL-2Rα Sushi domain. ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG (SEQ ID NO: 182)

[0105] In various embodiments, the PD1 Ab-IL-2 VitoKine contains IL-2RαSushi (SEQ ID NO: 182) as a masking moiety to mask IL-2 (including IL-2 variant) activity. Wild-type IL-2Rα binds to IL-2 with a moderate affinity of 30 nM, but there is still a possibility that IL-2Rα will not dissociate upon linker cleavage. Association between cleaved IL-2Rα and IL-2 may reduce the activity of IL-2 and / or tip the balance of T cell subpopulations toward undesirable outcomes. When affinity-reducing mutations are introduced into IL-2RαSushi, the IL-2Rα sushi domain is likely to dissociate from IL-2 upon linker cleavage. In various embodiments, the hidden partial domain in PD1 Ab-IL-2 VitoKine is an IL-2RαSushi variant containing an IL-2 binding-weakening mutation, e.g., R36A, K38E, L42G, or Y43A, or any combination of substitutions. In various embodiments, the IL-2RαSushi variant can effectively hide the activity of the IL-2 partial domain despite having reduced affinity for IL-2. In various embodiments, the IL-2RαSushi variant is expected to dissociate and diffuse from IL-2 upon linker cleavage due to its reduced affinity for IL-2.

[0106] In various embodiments, the PD1 Ab-IL-2 VitoKine constructs of the present invention contain a cryptic partial domain that is one of the IL-2Rα Sushi domain variants comprising the amino acid sequence set forth in SEQ ID NOs: 183-185.

[0107] L1 and L2 linkers in PD1 Ab-IL-2 VitoKine Cleavable Linker Cleavable linker, or linker sensitive to disease-related enzymes, may contain a moiety, such as a protein substrate, that has the ability to be specifically cleaved by proteases that are present at elevated levels in diseased tissues compared to non-diseased tissues.The literature contains multiple reports on the increased levels of enzymes with known substrates in various types of cancer, such as solid tumors.See, for example, La Rocca et al., Brit.J.Cancer 90:1414-1421 and Ducry et al., Bioconjug.Chem.21:5-13,2010 (each of which is incorporated herein by reference in its entirety). In various embodiments, the protease capable of cleaving the protease-cleavable linker is selected from the group consisting of metalloproteinases, e.g., matrix metalloproteinases (MMPs) 1-28, serine proteases, e.g., urokinase-type plasminogen activator (uPA) and matriptase, cysteine ​​proteases, e.g., legumain, aspartic acid proteases, and cathepsin proteases. Exemplary proteases are provided in Table 2: TIFF2025531804000002.tif217170

[0108] Exemplary protease substrate peptide sequences that can be used as protease-cleavable linkers, with or without a peptide spacer, are provided in Table 3: TIFF2025531804000003.tif148170

[0109] In various embodiments, the protease is MMP-9 or MMP-2. In further specific embodiments, the protease is matriptase. In further specific embodiments, the protease is MMP-14. In further specific embodiments, the protease is legumain. In various embodiments, the protease-cleavable linker may contain two or more protease substrate sequences. In various embodiments, the proteases are MMP-2 / MMP-9 and matriptase. In various embodiments, the protease-cleavable linker comprises the protease recognition sequence "GPLGMLSQ" (SEQ ID NO: 61). In various embodiments, the protease-cleavable linker comprises the protease recognition sequence "SGRSENIRTA" (SEQ ID NO: 60). In various embodiments, the protease-cleavable linker comprises the protease recognition sequence "GPTNKVR" (SEQ ID NO: 69). In various embodiments, the protease-cleavable linker comprises the protease recognition sequence "PMAKK" (SEQ ID NO: 74). In various embodiments, the protease-cleavable linker comprises the protease recognition sequence "GPLGMLSQPMAKK" (SEQ ID NO: 76). In various embodiments, the protease-cleavable linker comprises the protease recognition sequence "PMAKKGPLGMLSQ" (SEQ ID NO: 77).

[0110] In various embodiments, peptide spacers may be incorporated on either side of the protease-cleavable sequence, adjacent to both sides of the protease-cleavable sequence, or as a non-cleavable linker without a protease substrate site. The peptide spacer serves to position the cleavable linker, making it more accessible to the enzyme responsible for cleavage. The length and composition of the peptide spacer can be fine-tuned to balance accessibility for enzymatic cleavage with the spatial constraints required to reversibly conceal the D2 domain so that it does not exert its biological activity. The peptide spacer may contain 1 to 100 amino acids. Suitable peptide spacers are known in the art and include, but are not limited to, peptide linkers containing flexible amino acid residues, such as glycine and serine. In various embodiments, the peptide spacer can contain 1 to 12 amino acids including the motifs G, S, GSGG (SEQ ID NO: 104), GGSS (SEQ ID NO: 105), GSGS (SEQ ID NO: 109), GSGSGS (SEQ ID NO: 110), GSGSGSGS (SEQ ID NO: 111), GSGSGSGSGS (SEQ ID NO: 112), or GSGSGSGSGSGS (SEQ ID NO: 113). In other embodiments, the peptide spacer can contain 1 to 12 amino acids including the motifs G, S, GSGG (SEQ ID NO: 104), GGSS (SEQ ID NO: 105), GSGS (SEQ ID NO: 109), GSGSGS (SEQ ID NO: 110), GSGSGSGSGS (SEQ ID NO: 111), GSGSGSGSGS (SEQ ID NO: 112), or GSGSGSGSGSGS (SEQ ID NO: 113). n (where n is an integer from 1 to 10). In other embodiments, the peptide spacer may also contain amino acids other than glycine and serine. The peptide spacer is stable under physiological conditions as well as at disease sites, such as cancer sites.

[0111] Exemplary protease-cleavable linkers having a peptide spacer adjacent to a protease substrate peptide (underlined) are provided in Table 4: TIFF2025531804000004.tif111170

[0112] Non-cleavable linkers Non-cleavable linkers provide a covalent linkage between protein domains as well as additional structural and / or spatial flexibility. As known in the art, peptide linkers containing flexible amino acid residues, such as glycine and serine, can be used as non-cleavable linkers. In various embodiments, the non-cleavable linker may contain 1 to 100 amino acids. In various embodiments, the spacer can contain the motif GSGG (SEQ ID NO: 104), GGSS (SEQ ID NO: 105), GSGS (SEQ ID NO: 109), GSGSGS (SEQ ID NO: 110), GSGSGSGS (SEQ ID NO: 111), GSGSGSGSGS (SEQ ID NO: 112), or GSGSGSGSGSGS (SEQ ID NO: 113). In other embodiments, the spacer can contain the motif (GGGGS) (SEQ ID NO: 106). n (where n is an integer from 1 to 10). In other embodiments, the linker may also contain amino acids other than glycine and serine. In another embodiment, the non-cleavable linker may be a simple chemical bond, for example, an amide bond (e.g., by chemical conjugation of PEG). The non-cleavable linker is stable under physiological conditions as well as at disease sites, for example, at cancer sites.

[0113] Exemplary non-cleavable linkers are provided in Table 5: TIFF2025531804000005.tif112170

[0114] Combinations of cleavable and non-cleavable linkers In various embodiments, the L1 and L2 linkers can both be cleavable or a combination of cleavable and non-cleavable linkers, resulting in different forms of the active portion of the IL-2 domain to meet specific therapeutic objectives, optimize the risk-to-benefit ratio, or match diverse properties of the cytokine. Exemplary active forms released by linker cleavage are depicted in Figure 2. Active Form 1, derived from cleavage of the L1 linker, and Active Form 3, derived from cleavage of the L1 and L2 linkers, are both short-acting cytokines due to their release from the targeting antibody after proteolysis. The presence or absence of a cryptic domain results in distinct activities for these two active forms in the local environment. After acting locally, the short-acting active form can be rapidly cleared from the systemic circulation, leading to reduced toxicity. In contrast, Active Form 2, derived from cleavage of the L2 linker (depicted in Figure 2), is functionally fully restored IL-2 fused to the PD1 Ab at or near the disease site. This active form has the ability to cis-activate IL-2R signaling in PD1-expressing T cells at or near the site of disease, synergistically enhancing the two pathways and boosting anti-cancer immune responses while minimizing systemic toxicity.

[0115] Polynucleotides In another aspect, the present disclosure provides an isolated nucleic acid molecule comprising a polynucleotide of an IL-2, IL-2 variant, IL-2Rα, IL-2Rα variant, PD1-blocking antibody, antibody fragment, PD1 Ab-IL-2 VitoKine construct, or PD1-targeted IL-2 immunocytokine of the present disclosure. The subsequent paragraphs in this subsection, "Polynucleotides," utilize a PD1-targeted IL-2 VitoKine (VitoKine) construct as an illustrative example, but these concepts are equally applicable to the PD1-targeted IL-2 immunocytokines of the present invention.

[0116] The subject nucleic acids may be single-stranded or double-stranded. Such nucleic acids may be DNA or RNA molecules. DNA includes, for example, cDNA, genomic DNA, synthetic DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA encoding VitoKine constructs can be obtained from genomic libraries available for many species. Synthetic DNA can be obtained by chemical synthesis of overlapping oligonucleotide fragments followed by assembly of the fragments to reconstitute part or all of the coding region and flanking sequences. RNA can also be obtained from prokaryotic expression vectors that direct high-level synthesis of mRNA, such as vectors using a T7 promoter and RNA polymerase. DNA molecules of the present disclosure include full-length genes as well as polynucleotides and fragments thereof. Full-length genes may also include sequences encoding an N-terminal signal sequence. Such nucleic acids can be used, for example, in methods for producing novel VitoKine constructs.

[0117] In various embodiments, the isolated nucleic acid molecule comprises a polynucleotide described herein and further comprises a polynucleotide encoding at least one heterologous protein described herein, hi various embodiments, the nucleic acid molecule further comprises a polynucleotide encoding a linker or hinge linker described herein.

[0118] In various embodiments, the recombinant nucleic acid of the present disclosure may be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory sequences are art-recognized and are selected to direct expression of the VitoKine construct. Thus, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosomal binding site, transcriptional start and stop sequences, translational start and stop sequences, and an enhancer or activator sequence. Constitutive or inducible promoters, as known in the art, are contemplated by the present disclosure. The promoter may be either a naturally occurring promoter or a hybrid promoter that combines elements of more than one promoter. The expression construct may be present in the cell on an episome, such as a plasmid, or the expression construct may be inserted into a chromosome. In various embodiments, the expression vector contains a selectable marker gene to allow for the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

[0119] In another embodiment of the present disclosure, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding a VitoKine construct and operably linked to at least one regulatory sequence. The term "expression vector" refers to a plasmid, phage, virus, or vector for expressing a polypeptide from a polynucleotide sequence. Suitable vectors for expression in host cells are readily available, and nucleic acid molecules are inserted into the vector using standard recombinant DNA techniques. Such vectors can contain a variety of expression control sequences that control the expression of DNA sequences when operably linked, and the expression control sequences can be used in these vectors to express DNA sequences encoding VitoKine constructs. Useful expression control sequences include, for example, the SV40 early and late promoters, the tet promoter, the adenovirus or cytomegalovirus immediate-early promoter, the RSV promoter, the lac system, the trp system, the TAC or TRC system, the T7 promoter, whose expression is directed by T7 RNA polymerase, the phage lambda major operator and promoter region, the control region for the fd coat protein, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter for acid phosphatase (e.g., PhoS), the promoter for yeast a-mating factor, the polyhedron promoter of baculovirus systems, and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof. It should be understood that the design of the expression vector can depend on factors such as the choice of the host cell to be transformed and / or the type of protein desired to be expressed. Additionally, the copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. Exemplary expression vectors suitable for expression of VitoKine are pDSRa containing the VitoKine polynucleotide, and derivatives thereof, as well as any additional suitable vectors known in the art or described below.

[0120] The recombinant nucleic acids of the present disclosure can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either or both prokaryotic cells, eukaryotic cells (yeast, avian, insect, or mammalian cells). Expression vehicles for producing recombinant VitoKine constructs include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.

[0121] Some mammalian expression vectors contain both prokaryotic sequences to facilitate propagation of the vector in bacteria and one or more eukaryotic transcription units that are expressed in eukaryotic cells. pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses, such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205), can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems. Various methods used in preparing plasmids and transforming host organisms are well known in the art. For general recombinant procedures, as well as other suitable expression systems for both prokaryotic and eukaryotic cells, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some cases, it may be desirable to express recombinant polypeptides using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (e.g., pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (e.g., pAcUW1), and pBlueBac-derived vectors (e.g., the B-gal-containing pBlueBac III).

[0122] In various embodiments, vectors are designed for production of a subject VitoKine construct in Chinese hamster ovary (CHO) cells or human embryonic kidney 293 (HEK293) cells, such as the Pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wis.) As will be apparent, a subject genetic construct can be used to direct expression of a subject VitoKine construct in cells propagated in culture to produce proteins, including fusion or variant proteins, for example, for purification.

[0123] The present disclosure also relates to host cells transfected with a recombinant gene comprising a nucleotide sequence encoding the amino acid sequence for one or more of the subject VitoKine constructs. The host cell may be any prokaryotic or eukaryotic cell. For example, the VitoKine constructs of the present disclosure may be expressed in bacterial cells, such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art, such as CHO cells or HEK293 cells.

[0124] Thus, the present disclosure further relates to methods for producing the subject VitoKine constructs. For example, host cells transfected with an expression vector encoding a VitoKine construct can be cultured under appropriate conditions to allow expression of the VitoKine construct to occur. The VitoKine construct may be secreted and isolated from a mixture of cells and medium containing the VitoKine construct. Alternatively, the VitoKine construct may be retained in the cytoplasm or in a membrane fraction, and the cells may be harvested, lysed, and the protein isolated. Cell cultures include host cells, medium, and other by-products. Suitable media for cell culture are well known in the art.

[0125] The polypeptides and proteins of the present disclosure can be purified according to protein purification techniques well known to those skilled in the art. These techniques involve, at one level, crude fractionation of protein and non-protein fractions. Once the peptide polypeptide has been separated from other proteins, the peptide or polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). The terms "isolated polypeptide" or "purified polypeptide," as used herein, are intended to refer to a composition isolable from other components, in which the polypeptide has been purified to any degree relative to its naturally obtainable state. A purified polypeptide therefore also refers to a polypeptide that has been freed from the environment in which the polypeptide may naturally occur. Generally, "purified" refers to a polypeptide composition that has been subjected to fractionation to remove various other components, wherein the composition substantially retains its expressed biological activity. When the term "substantially purified" is used, this designation refers to a peptide or polypeptide composition in which the polypeptide or peptide forms the major component of the composition, e.g., comprises about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the protein in the composition.

[0126] Various techniques suitable for use in purification are well known to those skilled in the art.These include, for example, ammonium sulfate, PEG, and antibody (immunoprecipitation), or heat denaturation followed by centrifugation precipitation; chromatography, for example, affinity chromatography (protein A column), ion exchange, gel filtration, reverse phase, hydroxylapatite, hydrophobic interaction chromatography; isoelectric focusing; gel electrophoresis; and combinations of these techniques.As is generally known in the art, the order of carrying out various purification steps may be changed, or certain steps may be omitted, and still be considered to result in a suitable method for preparing substantially purified polypeptide.

[0127] Pharmaceutical Composition The subsequent paragraphs of this subsection, "Pharmaceutical Compositions," utilize PD1-targeted IL-2 VitoKine (VitoKine) constructs as an illustrative example, but these concepts are equally applicable to the PD1-targeted IL-2 immunocytokines of the present invention.

[0128] In another aspect, the present disclosure provides a pharmaceutical composition comprising a VitoKine construct mixed with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known and understood by those skilled in the art and have been described extensively (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company, 1990). Pharmaceutically acceptable carriers may be included for purposes such as modifying, maintaining, or preserving the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or permeability of the composition. Such pharmaceutical compositions may affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the polypeptide.Suitable pharmaceutically acceptable carriers include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, other organic acids); bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants; flavoring agents and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); lolidone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides (preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.

[0129] The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, saline solution, or artificial cerebrospinal fluid, optionally supplemented with other ingredients common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5 or acetate buffer at about pH 4.0-5.5, which may further contain sorbitol or a suitable substitute. In one embodiment of the present disclosure, the composition may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulating agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or aqueous solution. Additionally, therapeutic compositions may be formulated as lyophilizates using appropriate excipients, such as sucrose. The optimal pharmaceutical composition can be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage.

[0130] When parenteral administration is contemplated, the therapeutic pharmaceutical composition may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired VitoKine construct in a pharmaceutically acceptable medium. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the polypeptide is formulated as a sterile, isotonic solution, properly preserved. In various embodiments, pharmaceutical preparations suitable for injectable administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Additionally, suspensions of the active compound may be prepared as appropriate oily injection suspensions. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound and allow for the preparation of highly concentrated solutions.

[0131] In various embodiments, therapeutic pharmaceutical compositions may be formulated for targeted delivery using colloidal dispersion systems.Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.Liposome targeting can also be based on, for example, organ specificity, cell specificity, and organelle specificity, which is known in the art.

[0132] In various embodiments, it is envisaged that pharmaceutical compositions are orally administered.The pharmaceutical compositions administered in this manner can be formulated with or without the carriers that are commonly used in the preparation of solid dosage forms, such as tablets and capsules.In the solid dosage forms for oral administration (such as capsules, tablets, pills, dragees, powders and granules), one or more therapeutic compounds of the present disclosure can be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as acacia. The pharmaceutical compositions may be mixed with any of the following: (1) agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (2) dissolution retardants, such as paraffin; (3) absorption enhancers, such as quaternary ammonium compounds; (4) wetting agents, such as cetyl alcohol and glycerol monostearate; (5) absorbents, such as kaolin and bentonite clay; (6) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (7) coloring agents. For capsules, tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft- and hard-filled gelatin capsules, using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions can also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, coloring agents, fragrances, and preservatives.

[0133] In various embodiments, topical administration of the pharmaceutical composition to either the skin or mucous membranes is contemplated. Topical formulations may further include one or more of a variety of agents known to be effective as skin or stratum corneum permeation enhancers. Examples of these are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents may be included to make the formulation cosmetically acceptable. Examples of these are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surfactants. Keratolytic agents, such as those known to those skilled in the art, may also be included. Examples include salicylic acid and sulfur. Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives, buffers, or propellants. The ointments, pastes, creams and gels may contain, in addition to the subject compounds of the present disclosure (e.g., VitoKine constructs), excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0134] Additional pharmaceutical compositions contemplated for use herein include formulations involving polypeptides in sustained- or controlled-delivery formulations. In various embodiments, pharmaceutical compositions may be formulated as slow-release hydrogels, nanoparticles, or incorporated into oncolytic viruses. Methods for preparing such nanoparticles include, for example, encapsulation in nanoparticles composed of polymers with hydrophobic backbones and hydrophilic branches as drug carriers, encapsulation in microparticles, insertion into liposomes in emulsions, and conjugation to other molecules. Examples of nanoparticles include mucoadhesive nanoparticles coated with chitosan and carbopol (Takeuchi et al., Adv. Drug Deliv. Rev. 47(1):39-54, 2001) and nanoparticles containing the charged combination polyester, poly(2-sulfobutyl-vinyl alcohol) and poly(D,L-lactic-co-glycolic acid) (Jung et al., Eur. J. Pharm. Biopharm. 50(1):147-160, 2000). Albumin-based nanoparticle compositions have been developed as a drug delivery system for delivering hydrophobic drugs, such as taxanes.See, for example, United States Patent No. 5,916,596; United States Patent No. 6,506,405; United States Patent No. 6,749,868; United States Patent No. 6,537,579; United States Patent No. 7,820,788; and United States Patent No. 7,923,536.Abraxane (registered trademark), an albumin-stabilized nanoparticle formulation of paclitaxel, was approved in the United States in 2005 and subsequently in various other countries for the treatment of metastatic breast cancer.

[0135] Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art.

[0136] The effective amount of a pharmaceutical composition used therapeutically depends, for example, on the therapeutic context and purpose. Those skilled in the art will appreciate that appropriate dosage levels for treatment will therefore vary, in part, depending on the molecule being delivered, the condition for which the polypeptide is being used, the route of administration, and the patient's size (body weight, body surface, or organ size) and condition (age and general health). Thus, clinicians may titrate the dosage and modify the route of administration to obtain the optimal therapeutic effect. Typical dosages may range from about 0.0001 mg / kg to about 100 mg / kg or more, depending on the factors discussed above. Polypeptide compositions may be injected or administered preferably intravenously. Long-acting pharmaceutical compositions may be administered every 3-4 days, weekly, biweekly, triweekly, monthly, or for even longer durations, depending on the half-life and clearance rate of the particular formulation. The frequency of dosing depends on the pharmacokinetic parameters of the polypeptide in the formulation used. Typically, the composition is administered until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as multiple doses (of the same or different concentrations / dosages) over time, or as a continuous infusion. Further refinement of the appropriate dosage is routinely performed. The appropriate dosage may be ascertained through the use of appropriate dose-response data.

[0137] The pharmaceutical composition may be administered according to known methods, for example, orally, via injection, intravenously, intraperitoneally, intratumorally, intracerebrally (intraparenchymally), intraventricularly, intramuscularly, intraocularly, intraarterially, intraportally, intralesionally, intramedullary, intrathecally, intraventricularly, intravesically, transdermally, subcutaneously, or intraperitoneally; as well as intranasally, intestinal, topically, sublingually, urethrally, vaginally, or rectally, by sustained-release system, or by implantation device. If desired, the composition may be administered by bolus injection or continuously by infusion, or by implantation device. Alternatively, or additionally, the composition may be administered locally via implantation of a membrane, sponge, or another suitable material into which the desired molecule has been absorbed or encapsulated. When an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.

[0138] therapeutic use The subsequent paragraphs of this subsection, "Therapeutic Uses," utilize PD1-targeted IL-2 VitoKine (VitoKine) constructs as an illustrative example, but these concepts are equally applicable to the PD1-targeted IL-2 immunocytokines of the present invention.

[0139] The present disclosure provides a method for treating cancer cells in a subject, comprising administering to the subject a therapeutically effective amount (as monotherapy or in a combination treatment regimen) of a VitoKine construct of the present disclosure in a pharmaceutically acceptable carrier, wherein such administration inhibits the growth and / or proliferation of cancer cells. In particular, the VitoKine construct of the present disclosure is useful in treating disorders characterized as cancer. Such disorders include, but are not limited to, solid tumors, such as cancers of the breast, respiratory tract, brain, reproductive organs, gastrointestinal tract, urinary tract, eye, liver, skin, head and neck, thyroid, and parathyroid glands, and their distant metastases, lymphoma, sarcoma, multiple myeloma, and leukemia. Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ. Examples of cancers of the respiratory tract include, but are not limited to, small cell and non-small cell lung cancer, as well as bronchial adenoma and pulmonary pleuroblastoma. Examples of brain cancers include, but are not limited to, brainstem and hypothalamic glioma, cerebellar and cerebral astrocytoma, neuroblastoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumors. Tumors of the male reproductive organs include, but are not limited to, prostate and testicular cancer. Tumors of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as uterine sarcoma. Tumors of the gastrointestinal tract include, but are not limited to, anal, colon, colorectal, esophageal, gallbladder, stomach, liver, breast, pancreatic, rectal, small intestine, and salivary gland cancer. Tumors of the urinary tract include, but are not limited to, bladder, penile, kidney, renal pelvis, ureter, and urethral cancer. Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma. Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular-cholangiocarcinoma. Skin cancer includes, but is not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head and neck cancer includes, but is not limited to, nasopharyngeal carcinoma and lip cancer. Lymphoma includes, but is not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and lymphoma of the central nervous system.Sarcomas include, but are not limited to, sarcoma of soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

[0140] In various embodiments, VitoKine constructs may be used as single agents for the treatment of all types of cancer, including but not limited to non-small cell lung, small cell lung, melanoma, renal cell carcinoma, urothelial, liver, breast, pancreatic, colorectal, gastric, prostate, and sarcoma.

[0141] A "therapeutically effective amount" or "therapeutically effective dose" refers to that amount of a therapeutic agent being administered that will relieve to some extent one or more of the symptoms of the disorder being treated.

[0142] The therapeutically effective dose is IC 50 The dose can be estimated initially from cell culture assays by determining the IC (half maximal inhibitory concentration). The dose can then be calculated using the IC as determined in cell culture. 50 The compound can be formulated in animal models to achieve a circulating plasma concentration range including the range of 0.1 to 1.5 mg / kg of circulating plasma. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by HPLC. The exact composition, route of administration, and dosage can be chosen by the individual physician in consideration of the subject's condition.

[0143] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, or several divided doses (multiple or repeated or maintenance) can be administered over time, and the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present disclosure are primarily dictated by the unique characteristics of the antibody and the specific therapeutic or prophylactic effect to be achieved.

[0144] Therefore, those skilled in the art will understand based on the disclosure provided herein that dosage and dosage regimen will be adjusted according to the method well known in the therapeutic field.That is, the maximum acceptable dosage can be easily established, and the effective amount that provides detectable therapeutic benefit to the subject can also be determined, as well as the time requirement for administering each agent to provide detectable therapeutic benefit to the subject.Therefore, although certain dosage and dosage regimen are exemplified herein, these examples do not limit the dosage and dosage regimen that can be provided to the subject in the implementation of the present disclosure.

[0145] It should be noted that dosage values ​​may vary with the type and severity of the condition to be alleviated and may include single or multiple doses. For any particular subject, specific dosage regimens should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and it should be further understood that the dosage ranges described herein are merely exemplary and are not intended to limit the scope or implementation of the claimed compositions. Furthermore, dosage regimens using the compositions of the present disclosure may be based on various factors, including the type of disease, the subject's age, weight, sex, medical condition, severity of the condition, and route of administration. Thus, dosage regimens can vary widely but can be routinely determined using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects, such as toxic effects and / or laboratory values. Therefore, the present disclosure encompasses intra-subject dose escalation as determined by those skilled in the art. It will be appreciated that determination of appropriate dosages and regimens is well known in the relevant art and could be accomplished by one of ordinary skill in the art once provided with the teachings disclosed herein.

[0146] Exemplary, non-limiting daily dosage ranges for a therapeutically or prophylactically effective amount of VitoKine, or a VitoKine variant of the present disclosure, are 0.0001-100 mg / kg, 0.0001-90 mg / kg, 0.0001-80 mg / kg, 0.0001-70 mg / kg, 0.0001-60 mg / kg, 0.0001-50 mg / kg, 0.0001-40 mg / kg, 0.0001-30 mg / kg, 0.0001- 20mg / kg, 0.0001~10mg / kg, 0.0001~5mg / kg, 0.0001~4mg / kg, 0.0001~3mg / kg, 0.0001~2mg / kg, 0.0001~1mg / kg, 0.001 ~50mg / kg, 0.001~40mg / kg, 0.001~30mg / kg, 0.001~20mg / kg, 0.001~10mg / kg, 0.001~5mg / kg, 0.001~4mg / kg, 0.001~3 mg / kg, 0.001~2mg / kg, 0.001~1mg / kg, 0.010~50mg / kg, 0.010~40mg / kg, 0.010~30mg / kg, 0.010~20mg / kg, 0.010~10m g / kg, 0.010~5mg / kg, 0.010~4mg / kg, 0.010~3mg / kg, 0.010~2mg / kg, 0.010~1mg / kg, 0.1~50mg / kg, 0.1~40mg / kg, 0.1~ The dosage may be 30 mg / kg, 0.1-20 mg / kg, 0.1-10 mg / kg, 0.1-5 mg / kg, 0.1-4 mg / kg, 0.1-3 mg / kg, 0.1-2 mg / kg, 0.1-1 mg / kg, 1-50 mg / kg, 1-40 mg / kg, 1-30 mg / kg, 1-20 mg / kg, 1-10 mg / kg, 1-5 mg / kg, 1-4 mg / kg, 1-3 mg / kg, 1-2 mg / kg, or 1-1 mg / kg body weight. It should be noted that dosage values ​​may vary with the type and severity of the condition to be alleviated. It should be further understood that for any particular subject, specific dosage regimens will be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[0147] The toxicity and therapeutic index of the pharmaceutical compositions of the present disclosure are, for example, LD 50 (a dose lethal to 50% of the population) and ED 50 The dose that is therapeutically effective in 50% of a population can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutically effective doses is the therapeutic index, which is the ratio LD 50 / ED 50 Compositions that exhibit large therapeutic indices are generally preferred.

[0148] The dosage frequency of administering VitoKine construct pharmaceutical composition depends on the nature of treatment and the specific disease being treated.Subjects can be treated at regular intervals, for example, weekly or monthly, until the desired therapeutic result is achieved.Exemplary dosage frequencies include, but are not limited to, once a week without interruption; once every two weeks; once every two weeks; once every three weeks; once a week for two weeks without interruption, then monthly; once a week for three weeks without interruption, then monthly; once a month; once every two months; once every three months; once every four months; once every five months; or once every six months, or yearly.

[0149] Combination treatment The subsequent paragraphs of this subsection, "Combination Therapy," utilize PD1-targeted IL-2 VitoKine (VitoKine) constructs as an illustrative example, but these concepts are equally applicable to the PD1-targeted IL-2 immunocytokines of the present invention.

[0150] As used herein, the terms "co-administration," "co-administered," and "in combination with," referring to a VitoKine construct of the present disclosure and one or more other therapeutic agents, are intended to mean, and refer to and include, the simultaneous administration of a VitoKine construct of the present disclosure and a combination of therapeutic agents to a subject in need of treatment, where such components are formulated together in a single dosage form that releases said components at substantially the same time in said subject; and the substantially simultaneous administration of a VitoKine construct of the present disclosure and a combination of therapeutic agents to a subject in need of treatment, where such components are formulated separately from one another in separate dosage forms that are taken by said subject at substantially the same time and that release said components at substantially the same time in said subject. the sequential administration of a combination of a VitoKine construct of the present disclosure and a therapeutic agent to a subject in need of treatment, where such components are formulated apart from one another to be in separate dosage forms taken by the subject at successive times with a significant time interval between each administration, and where the components are released to the subject at substantially different times; and the sequential administration of a combination of a VitoKine construct of the present disclosure and a therapeutic agent to a subject in need of treatment, where such components are formulated together to be in a single dosage form that releases the components in a controlled manner, where they are released to the subject at the same and / or different times, concurrently, sequentially, and / or overlappingly, and where each portion may be administered by the same or different routes.

[0151] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention in combination with a second treatment, including but not limited to immunotherapy, cytotoxic chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, and stem cell transplantation.For example, such a method can be used in preventive cancer prevention, preventing cancer recurrence and metastasis after surgery, and as an adjuvant for other conventional cancer treatments.The present disclosure recognizes that the effectiveness of conventional cancer treatments (such as chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through the use of the combined methods described herein.

[0152] A wide range of conventional compounds have been shown to have anti-neoplastic activity.These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastasis and further growth, or reduce the number of malignant T cells in leukemia or myeloid malignant tumors.Although chemotherapy has been effective in treating various types of malignant tumors, many anti-neoplastic compounds induce undesirable side effects.It has been shown that when two or more different treatments are combined, the treatments can function synergistically and allow the dosage of each treatment to be reduced, thereby reducing the harmful side effects caused by each compound at higher dosages.In other cases, malignant tumors that are refractory to treatment can respond to the combined treatment of two or more different treatments.

[0153] In various embodiments, a second anti-cancer agent, e.g., a chemotherapeutic agent, is administered to the patient. A list of exemplary chemotherapeutic agents includes daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, bendamustine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin, These include, but are not limited to, combinations of agents such as, but not limited to, carboplatin, oxaliplatin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fludarabine, ifosfamide, hydroxyurea taxanes (e.g., paclitaxel and docetaxel) and / or anthracycline antibiotics, as well as DA-EPOCH, CHOP, CVP, or FOLFOX. In various embodiments, the dosage of such chemotherapeutic agents is about 10 mg / m 2 , 20 mg / m 2 , 30 mg / m 2 , 40 mg / m 2 , 50 mg / m 2 , 60 mg / m 2 , 75 mg / m 2 , 80 mg / m 2 , 90 mg / m 2 , 100 mg / m 2 , 120 mg / m 2 , 150 mg / m 2 , 175 mg / m 2 , 200 mg / m 2 , 210 mg / m 2 , 220 mg / m 2 , 230 mg / m 2 , 240 mg / m 2 , 250 mg / m 2 , 260 mg / m 2 , and 300 mg / m 2 This includes, but is not limited to, any of the following:

[0154] In various embodiments, the combination treatment methods of the present disclosure include treatments using depleting antibodies against specific tumor antigens; treatments using antibody-drug conjugates; treatments using antibodies against specific tumor antigens, such as CTLA-4, PDL-1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, SIRPα, CD47, GITR, ICOS, CD27, Siglec 7, Siglec 8, Siglec 9, Siglec treatments using agonist, antagonist, or blocking antibodies against costimulatory or co-inhibitory molecules (immune checkpoints), including, but not limited to, antibodies against VISTA, CD276, CD272, TIM-3, and B7-H4; treatments using bispecific T cell engaging antibodies (BiTEs®), such as blinatumomab; treatments involving the administration of biological response modifiers, such as IL-7, IL-10, IL-12, IL-15, IL-21, IL-22, GM-CSF, IFN-α, IFN-β, IFN-γ, TGF-β antagonists, or TGF-β traps; treatments using therapeutic vaccines, including, but not limited to, oncolytic viruses, such as T-vec, or therapeutic vaccines, such as sipuleucel-T; treatments using dendritic cell vaccines, or tumor antigen peptide or neoantigen vaccines; treatments using chimeric antigen receptor (CAR)-T cells; The combination treatment may further comprise administering to the subject a therapeutically effective amount of immunotherapy, including but not limited to, treatment using AR-NK cells; treatment using NK cells; treatment using iPS-induced NK cells; treatment using iPS-induced T cells; treatment using iPS-induced CAR-T or iPS-induced CAR-NK cells; treatment using tumor-infiltrating lymphocytes (TIL); treatment using adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR-T cells); treatment using TALL-104 cells; and treatment using immunostimulants, such as Toll-like receptor (TLR) agonists CpG, TLR7, TLR8, TLR9, and vaccines, such as Bacillus Calmette-Guerin (BCG), and imiquimod; the combination treatment provides increased effector cell killing of tumor cells, i.e., synergism exists between the VitoKine construct and the immunotherapy when administered in combination.

[0155] In various embodiments, the combination therapy involves simultaneous administration of the VitoKine construct and the second agent composition, either in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the VitoKine construct composition and the second agent composition are administered sequentially, i.e., the VitoKine construct composition is administered either before or after the administration of the second agent composition. In various embodiments, the administration of the VitoKine construct composition and the second agent composition is concurrent, i.e., the administration periods of the VitoKine construct composition and the second agent composition overlap with each other. In various embodiments, the administration of the VitoKine construct composition and the second agent composition is non-concurrent. For example, in various embodiments, the administration of the VitoKine construct composition is terminated before the administration of the second agent composition. In various embodiments, the administration of the second agent composition is terminated before the VitoKine construct composition is administered.

[0156] The following examples are given to more fully illustrate the present disclosure, but are not to be construed as limiting the scope of the invention. [Example]

[0157] Example 1 Sequence optimization of pembrolizumab variable domains The present invention aims to optimize the variable domain sequence of pembrolizumab to enhance its similarity score to human germline sequences, an indicator of "humanity." This enhancement could potentially reduce the risk of immunogenicity. Additionally, the inventors used human VH3 family germline sequences, which are less compatible with pembrolizumab but are more prevalent and behave better as alternative acceptor frameworks. This was done with the goal of improving the biophysical properties of the resulting humanized antibody, ensuring full activity, and enhancing its sequence humanity.

[0158] Pembrolizumab was humanized by CDR grafting technology using the most homologous human antibody sequence available in the RCSB Protein Data Bank as the acceptor human framework. The frameworks found in GenBank under accession numbers AB063829 (SEQ ID NO: 40) and M29469 (SEQ ID NO: 41) were used as the acceptor human frameworks for the heavy chain variable domain (VH) and light chain variable domain (VL), respectively (Carven GJ et al., U.S. Patent No. 8,354,509 B2). However, pembrolizumab only shares 79.6% sequence identity with the closest human germline IGHV1-2, according to a comparison of the variable region exons using the International Immunogenetics Information System (IMGT) DomainGapAlign tool (www.imgt.org). Similarity scores to human germline sequences were proposed as a definitional criterion for therapeutic antibodies by the WHO's International Nonproprietary Names (INN) group in 2014, presumably based on the concept that higher similarity may indicate reduced immunogenicity. The low similarity score, or "degree of humanness" (Abhinandan KR et al., J Mol Biol (2007) 369:852-62) of the heavy chain of pembrolizumab may be due to the low level of conservation between the mouse CDRs and their human sequence counterparts, the need to retain a few structurally important mouse framework residues to recapitulate antigen binding, and the conservation of unique somatic mutations in the human framework sequence AB063829.

[0159] To enhance the degree of humanization of pembrolizumab, certain CDR residues were targeted for replacement with equivalent residues from the closest human germline sequence. This method is referred to herein as CDR germlining. While avoiding CDR perturbations has traditionally been a central principle in humanized Ab design, only a limited number of CDR residues are involved in direct antigen interactions. Therefore, certain CDR residues can be replaced without impairing antibody activity. According to the Kabat numbering scheme, CDRs are defined as amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), 89-97 (CDR-L3), 31-35b (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3).

[0160] Regarding the CDR3 sequence, a portion of the light chain CDR3 (CDR-L3) and the entire heavy chain CDR3 (CDR-H3) were not part of the variable region exon V region of the germline sequence. Consequently, no human germline residues were available to replace the mouse CDR counterparts. Additionally, CDR3, especially CDR-H3, is highly variable and is crucial for antigen binding and functional activity, making it essential to preserve its conformation. Therefore, both the CDR-L3 (QHSRDLPLT; SEQ ID NO: 25) and CDR-H3 (RDYRFDMGFDY; SEQ ID NO: 33) of pembrolizumab were excluded from the CDR germlining process.

[0161] The CDR-L1 and CDR-L2 sequences of pembrolizumab were aligned to their counterparts from the closest human germline, IGKV3D-11 (GenBank accession # X17264; SEQ ID NO: 39). The alignment is shown in Table 6A. Similarly, the alignment of the CDR-H1 and CDR-H2 sequences of pembrolizumab with the closest human germline sequence, IGHV1-2 (GenBank accession # X62106; SEQ ID NO: 37), is shown in Table 6B. TIFF2025531804000006.tif100170

[0162] Several pembrolizumab CDR residues are directly involved in polar interactions with PD1, such as hydrogen bonds and salt bridges (Horita, S. et al. Sci. Rep (2016). 6:35297). Contact residues in or near CDR1 and CDR2 of both VL and VH are located in CDR-L1. L Ser28, L Tyr30, just before CDR-L2 L Tyr49 in CDR-L2 L Tyr53 in CDR-H1 H Tyr33, H Tyr35 in CDR-H2 H Asn52, H Ser53, H Asn54, H Thr57, H Asn58 (where superscript "L" denotes light chain and "H" denotes heavy chain). CDR residues not shown L With the exception of Tyr49, all antigen-interacting CDR residues listed above are in bold and italic in Tables 6A and 6B. Among the CDR residues that differ from the germline sequence are the CDR-L1 residues L Lys27, L His34, CDR-L2 residue L Leu54, L Glu55, CDR-H2 residue H Phe59, H Asn60, H Glu61, H Lys64, and H Asn65 (underlined in Tables 6A and 6B) was selected for CDR germlining, with the following amino acid substitutions, either individually or in combination: L K27Q, L H34A, L L54R, L E55A, H F59Y, H N60A, H E61Q, H K64Q, and HThey were replaced with their respective human germline equivalents with N65G. Other CDR residues were preserved to avoid any disruption of antigen-interacting residues.

[0163] For CDR-H1 (NYYMY; SEQ ID NO: 26), a single residue: H Only Asn31 is eligible for CDR germlining and is replaced with its equivalent residue in the human germline sequence, glycine. The other residues are involved in direct antigen interaction with PD1 ( H Tyr33 and H Tyr35), or conserved between mouse and human germline sequences ( H Tyr32 and H However, considering the short length of CDR-H1, which is only 5 amino acids, and the fact that two residues have direct interactions with the antigen, any amino acid changes could potentially disrupt the CDR conformation and affect the activity of the antibody. H Asn31 is unaltered throughout CDR germlining, and CDR-H1 is completely conserved.

[0164] In addition to the low level of conservation between the mouse CDRs and their corresponding human germline sequences, the pembrolizumab heavy chain framework (FR) also contains multiple non-germline residues, which arise due to the retention of unique somatic mutations in the acceptor framework sequence AB063829. H Val9, FR-H3 H Thr76, H Lys82a, H Gln83, H in Phe84 and FR-H4 H These somatic mutations, including Thr108, ​​are not believed to be structurally significant. H V9A, H T76S, H K82aS, H Q83R, H F84S,H The substitution with T108L leads to a significant improvement in the similarity score to the human germline sequence without disrupting the CDR conformation or altering the activity of the antibody.

[0165] Furthermore, we used IGHV3-23 (SEQ ID NO: 38) as an alternative acceptor framework to investigate whether utilizing a human acceptor framework with substantially lower sequence homology but superior biophysical attributes could enhance the biophysical properties of the resulting humanized antibody without compromising functional activity. IGHV3-23 belongs to the human antibody heavy chain germline VH3 family, the most common VH family in the human repertoire. It is also the most prevalent among commercially available human monoclonal antibodies and is widely recognized for its excellent drug-like properties. Considering that CDR conformation is highly sensitive to the chemical environment of the surrounding framework, a small number of structurally significant framework residues in pembrolizumab that differ from their VH3 germline family equivalents were selected for backmutation to their corresponding pembrolizumab equivalents. Furthermore, to improve the similarity score to the human germline sequence, several CDR-H2 residues were targeted for CDR germlining using IGHV3-23 CDR-H2 as a template.

[0166] The alignment of the CDR-H1 and CDR-H2 sequences of pembrolizumab with IGHV3-23 is shown in Table 6C. The six CDR-H2 residues: H Phe59, H Asn60, H Glu61, H Lys62, H Phe63, and H Asn65 (underlined in Table 6C; superscript "L" represents the light chain and "H" refers to the heavy chain) with the following amino acid substitution: H F59Y, H N60A, H E61D, H K62S, H F63V, andH It was selected for CDR germlining at N65G. CDR-H1 was excluded from the CDR germlining process for the reasons mentioned above. TIFF2025531804000007.tif53170

[0167] two framework residues, H Thr30 and H Arg94 is considered structurally significant and its corresponding germline equivalent, H Ser30 and H Lys94 was preserved without modification. Five additional IGHV3 framework residues that belong to the Vernier zone (U.S. Pat. Nos. 5,821,337 and 5,859,205) and may have structural significance: H Val48, H Ser49, H Ile69, H Arg71, and H Asn73 to their corresponding pembrolizumab residues, either individually or in combination, H Met48, H Gly49, H Leu69, H Thr71, and H The amino acid sequence was reverted to Ser73. The importance of specific framework amino acid residues was evaluated experimentally. The number of backmutations was minimized to ensure the highest similarity score to the germline sequence without negatively affecting antibody activity.

[0168] All optimized antibody sequences were expressed as full-length antibodies with a kappa light chain constant region containing the sequence set forth in SEQ ID NO: 34 and a modified IgG1 heavy chain constant region containing the sequence set forth in SEQ ID NO: 35. Table 7 lists the VL, VH, CDR-L1, CDR-L2, and CDR-H2 SEQ ID NOs for exemplary optimized PD1-blocking antibodies, along with a reference antibody (P-0734) containing the VL and VH sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 6, respectively. All antibodies of the invention contain the same CDR-L3 (SEQ ID NO: 25), CDR-H1 (SEQ ID NO: 26), and CDR-H3 (SEQ ID NO: 33). TIFF2025531804000008.tif151170

[0169] Example 2 Construction, production, and purification of optimized PD1-blocking antibodies All genes were codon-optimized for expression in mammalian cells and were synthesized and subsequently subcloned into their recipient mammalian expression vectors through the services of GenScript. Protein expression was driven by a CMV promoter, and a synthetic SV40 polyA signal sequence was placed at the 3' end of the coding sequence. A leader sequence was engineered at the N-terminus of the construct to ensure proper signal transduction and processing for secretion.

[0170] Antibodies were produced by co-transfecting vectors harboring the light and heavy chains in a 1:1 ratio in ExpiCHO cells (ThermoFisher) according to the manufacturer's instructions. On the day of transfection, ExpiCHO cells were grown at 6 x 10 in ExpiCHO™ Expression Medium (ThermoFisher). 6The cells were diluted to 1000 cells / mL. A total of 0.8 μg of DNA expression vector per mL of culture volume was mixed with cold OptiPRO™ medium (40 μL per mL of cell culture). After adding ExpiFectamine™ CHO reagent at 3.2 μL per mL of cell culture, the solution was gently mixed and subsequently incubated at room temperature for 5 minutes. The ExpiFectamine™ CHO / plasmid DNA complex was then gently transferred to the cells and incubated at 130 rpm in a 37°C shaker incubator with an 8% CO2 atmosphere. ExpiFectamine™ CHO Enhancer (6 μL per mL of cell culture) and ExpiCHO™ Feed (240 μL per mL of cell culture) were added to the flask with gentle swirling 18-22 hours after transfection. After 8 days of culture, the supernatant was collected for purification by centrifugation at 2200 rpm for 20 min, followed by sterile filtration using a 0.22 μm filter (Corning).

[0171] Secreted antibodies were purified from cell culture supernatants using Protein A affinity chromatography. The cell culture supernatant was loaded onto a MabSelect SuRe 5 mL column (Cytiva) equilibrated with 5 column volumes (CV) of phosphate-buffered saline (pH 7.2) (ThermoFisher). Unbound proteins were removed by washing with 5 CV of PBS (pH 7.2), and the target protein was eluted with 25 mM sodium citrate, 25 mM sodium chloride buffer (pH 3.2). The antibody solution was neutralized by adding 3% 1 M Tris buffer (pH 10.2), followed by concentration and buffer exchange into PBS (pH 7.2) using an Amicon® Ultra-15 Ultracel (Merck Millipore) with a 10 KDa MWCO.

[0172] The purity and molecular weight of the purified antibodies were analyzed by SDS-PAGE with and without reducing agents and then stained with Coomassie (Imperial™ protein stain, ThermoFisher). The SurePAGE™ Pre-Cast gel system (8-16% Bis-Tris, GenScript) was used according to the manufacturer's instructions. The aggregate content of the antibodies was analyzed on an Agilent 1200 high-performance liquid chromatography (HPLC) system. Samples were injected onto an AdvanceBio size-exclusion column (300 Å, 4.6 × 150 mm, 2.7 μm, LC column, Agilent) using 150 mM sodium phosphate buffer (pH 7.0) as the mobile phase at 25 °C.

[0173] The antibody concentration of purified protein samples was determined by measuring absorbance at 280 nm using a Nanodrop spectrophotometer (ThermoFisher) and dividing it by the molar extinction coefficient calculated based on the amino acid sequence. Endotoxin levels of purified protein samples were measured using Endosafe nexgen-PTS (Charles River) according to the manufacturer's instructions.

[0174] Example 3 Assays to evaluate the biological activity of optimized PD1-blocking antibodies The antibodies of the present invention were tested for their antigen-binding activity by well-known methods, such as enzyme-linked immunosorbent assay (ELISA). Briefly, Nunc Maxisorp plates (ThermoFisher) were coated with recombinant human PD1 protein in bicarbonate buffer (pH 9.4) (ThermoFisher) overnight at 4°C using 1 μg of antigen per well (100 μL / well). After triple washing with PBS / 0.05% Tween 20, the plates were incubated with SuperBlock (ThermoFisher) for 2 hours at room temperature to block nonspecific binding. PD1 antibodies serially diluted 3-fold in blocking buffer (PBS containing 1% bovine serum albumin) were added to the washed plates (100 μL / well) and incubated for 1 hour at room temperature. After another wash, the antibodies were detected by incubation with horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc antibody (ThermoFisher) diluted 1:5000 in blocking buffer (100 μL / well) for 1 hour at room temperature. After the final wash, 100 μL / well of TMB substrate (ThermoFisher) was added. The plate was sealed and incubated in the dark for 5-20 minutes. The reaction was stopped by adding 2N sulfuric acid (Ricca Chemical) (50 μL / well), and the absorbance was measured at 450 nm using a plate reader. Curves were plotted and the half-maximal effective concentration (EC) was calculated using GraphPad Prism software. 50 ) values ​​were calculated.

[0175] Additionally, HEK 293T cells (Crown Bioscience) stably expressing the human PD1 gene were used to determine the cell-based binding strength of the optimized PD1 antibodies by flow cytometry. After harvesting, HEK293-hPD1 cells were cultured at 1 × 10 in 96-well U-bottom plates. 5Cells were seeded at 100 μL per well and incubated with Fc block (1:50) for 20 minutes at 4°C and subsequently washed with FACS buffer (PBS, 1% FBS). Cells were then treated with 3-fold serial dilutions of each antibody in FACS buffer at concentrations ranging from 0.01 to 100 nM for 30 minutes at 4°C. Subsequently, cells were washed twice with FACS buffer to remove unbound molecules, and 40 μL of 1:100 diluted PE-labeled goat anti-human Fc secondary antibody (eBiosciences) was added to the cells. After a 30-minute incubation at 4°C and another double wash with FACS buffer, cell-bound antibodies were detected by flow cytometry (BD ACCURI-C6) using the PE-labeled secondary antibody, and EC was analyzed using GraphPad Prism software. 50 The value was calculated.

[0176] Furthermore, we measured potency in blocking PD1 interactions using a biologically relevant mechanism-based assay, the Promega luciferase reporter assay in a thaw-and-use format. Cell thawing and plating procedures were followed exactly as described in the manufacturer's protocol.

[0177] Briefly, one vial (0.5 mL) of PD-L1 aAPC / CHO-K1 cells was thawed and mixed with 14.5 mL of cell recovery medium (90% Ham's F12 / 10% FBS). Next, 100 μL of this cell suspension was added to the inner 60 wells of two 96-well flat-bottom assay plates, with the surrounding wells receiving 100 μL of cell recovery medium. After overnight incubation at 37°C and 5% CO2, the medium was discarded. The inner wells received 40 μL of 3-fold serially diluted compounds, and the surrounding wells received 80 μL of assay buffer (99% RPMI 1640 / 1% FBS). Subsequently, one vial (0.5 mL) of PD1 effector cells was thawed and mixed with 5.9 mL of assay buffer, and 40 μL of this mixture was added to the inner wells. After 6 hours of incubation at 37°C, 5% CO2, and 7 minutes of equilibration at room temperature, 80 μL of Bio-Glo™ reagent was added to all wells. The plate was then incubated for 10 minutes at room temperature with shaking. The resulting luminescence was measured using a luminescence plate reader (BioTek synergy h1).

[0178] Background was calculated by averaging the relative light units (RLU) of surrounding wells. Fold induction was determined by subtracting the background from the RLU of the antibody sample and dividing by the RLU of the control sample (no antibody) minus the background, or Fold induction = RLU (antibody - background) / (RLU (no antibody control - background). Finally, the EC 50 value was determined.

[0179] Example 4 Evaluation of PD1 antibodies containing germline modifications based on the closest human germline sequence First, we compared the efficacy of P-0734 with that of the pembrolizumab (PBL) biosimilar in blocking PD1 / PD-L1 interaction. P-0734 and the PBL biosimilar share identical variable domains, but they differ in their heavy chain constant regions. PBL contains an IgG4 constant chain (SEQ ID NO: 36) containing the S228P mutation, while P-0734 has an IgG1 constant chain (SEQ ID NO: 35) with L234A / L235A / G237A mutations to prevent Fc effector function. As shown in Figure 4, P-0734 and the PBL biosimilar were equally effective in blocking the interaction between PD1 and PD-L1. This result was expected and confirms that the ability to block PD1 is determined by the variable domain sequence, not by immunoglobulin class. P-0734 faithfully reproduced the efficacy of the PBL biosimilar in blocking the PD1 / PD-L1 interaction and is referred to herein as the reference antibody.

[0180] in CDR-L1 using antibodies with slightly different mutational contexts L In H34A and CDR-H2 H The impact of the F60Y CDR germline substitution was evaluated. These antibodies, P-1148, P-1150, P-1151, and P-1153, all had a CDR-L1 germline substitution of F60Y. L K27Q, CDR-L2 L L54R, L E55A and CDR-H2 H N60A, H E61Q, H K64Q, H P-1150 contains the germline substitution N65G. L P-1151 contains an H34A substitution and an additional H F59Y substitution, and P-1153 L H34A and H Table 8 provides a list of CDR germline substitutions for these exemplary PD1 blocking antibodies. TIFF2025531804000009.tif47170

[0181] As shown in Figures 5B & 5C and summarized in Table 9, CDR-L1 germline substitutions L The H34A is H Regardless of the presence (P-1151 vs. P-1153) or absence (P-1148 vs. P-1150) of the F59Y substitution, E max PD1 blockade efficacy (EC 50 ) consistently led to an approximately 3.5-fold reduction in H The impact of the F59Y germline substitution was similarly assessed and the data are shown in Figures 5B & 5C and summarized in Table 9. L regardless of the presence (P-1150 vs. P-1153) or absence (P-1148 vs. P-1151) of the H34A substitution. H The F59Y CDR germline substitution increases potency (EC 50 ; approximately 1.8-fold reduction) and signal (E max Compared to P-0734, the cumulative CDR germline substitutions in P-1153 resulted in a small but consistent reduction in both PD1 blockade potency (EC 50 ) and an almost 20-fold decrease in E max This ultimately led to a 50% reduction in L H34A and H Both substitutions, F59Y and F59Y, are considered deleterious in this particular framework, and the original CDR residues, L His34 and H Phe59 will be conserved.

[0182] However, despite significant differences in potency of blocking the PD1 / PD-L1 interaction, all four optimized PD1 antibodies and the reference antibody, P-0734, had EC values ​​close to 100 pM. 50 The binding strengths were almost identical with the values ​​(Fig. 5A and Table 9). TIFF2025531804000010.tif59170

[0183] This body of data suggested that mechanism-based functional assays have the ability to discern subtle changes in activity that are not detectable by ELISA binding assays. Therefore, the luciferase PD1 / PD-L1 reporter assay is used herein as the primary tool to characterize and rank PD1-blocking antibodies derived from pembrolizumab via germline substitutions. Derivative antibodies that maintain full functional activity are expected to exhibit the same in vivo efficacy as pembrolizumab.

[0184] CDR-L2 germline substitutions by comparing P-1127 and P-1129 L The potential negative effects of E55A were further evaluated. Both of these molecules contain nucleotides in the light chain CDRs. L K27Q and L Contains the K54E germline substitution, with the only sequence difference being an additional CDR-L2 substitution at P-1129, L As demonstrated in Figure 6A, P-1129 showed a slight but noticeable reduction in potency (EC 3.0 for P-1127 and P-1129, respectively). 50 = 0.39 nM and 0.58 nM) and E max showed a slight 10% decrease in L The E55A amino acid substitution was considered deleterious, and the original residue, L Glu55 will be conserved.

[0185] A total of six CDR germline substitutions, L K27Q, L L54R, H N60A, H E61Q, H K64Q, and H P-1174, harboring N65G, exhibited identical PD1 blocking activity to P-1127 and P-0734, with EC values ​​of 0.64 nM, 0.54 nM, and 0.67 nM for P-0734, P-1127, and P-1174, respectively.50 (Figure 6B). Additionally, P-1174 contains one CDR germline substitution, L P-1174 was derived from P-114 by eliminating E55A. When compared to P-0734, P-1174 exhibited higher potency than P-1148 (see Figures 5B & 6B). This portion of the data shows that the original CDR residues, L They further concurred in the conclusion that Glu55 should not be altered.

[0186] Besides the low conservation between the mouse CDRs and their human germline counterparts, multiple non-germline residues in the pembrolizumab VH framework also contributed to the low sequence similarity score with the germline. H Val9, FR-3 H Thr76, H Lys82a, H Gln83, H in Phe84 and FR-4 H These non-germline residues, including Thr108, ​​originating from conserved unique somatic mutations in the acceptor framework sequence, are not considered structurally significant. To further enhance the sequence similarity score or degree of similarity to the germline, these non-germline residues in the P-1174 framework were compared with their respective germline counterparts. H V9A, H T76S, H K82aS, H Q83R, H F84S, H As expected, P-1271 exhibited the same PD1 blocking activity as the reference antibody, P-0734 (Figure 6C), with EC values ​​of 0.66 nM for P-1271 and 0.70 for P-0734, respectively. 50 was the value.

[0187] In conclusion, CDR germline substitutions at P-1174 and P-1271, L K27Q, L L54R, H N60A, H E61Q,H K64Q, and H N65G enhanced the degree of humanization of the antibody sequence without compromising potency in blocking the PD1 / PD-L1 interaction. Six additional framework germline substitutions in P-1271 further increased the similarity score to the closest human germline sequence. Table 10 lists the germline substitutions and similarity scores to the closest human germline sequence of P-1174 and P-1271 compared to the reference antibody, P-0734. TIFF2025531804000011.tif67170

[0188] Example 5 Evaluation of PD1 antibodies containing germline modifications based on the more prevalent human germline family (VH3) The incorporation of framework germlining substitutions based on the human antibody heavy chain germline IGHV3-23 (SEQ ID NO: 38) was investigated to test whether antibody frameworks with substantially lower sequence homology but superior biophysical properties could enhance the drug-like properties of the resulting antibodies while fully retaining functional activity. Of the 33 framework germlining substitutions (Table 11A), five Vernier zone residues: H V48, H S49, H I69, H R71, and H The importance of N73, their respective pembrolizumab equivalents, individually or in combination, H V48M, H S49G, H I69L, H R71T, and H This was experimentally evaluated by backmutation to N73S. Additionally, six CDR-H2 residues, H F59Y, H N60A, H E61D, H K62S, H F63V, and HN65G was selected for CDR germline substitution with their corresponding residue in IGHV3-23. Table 11B provides a summary of VH3 germline substitutions in exemplary antibodies. TIFF2025531804000012.tif169170

[0189] Figure 7 depicts the PD1 blocking activity of P-1175 and P-1181, which differ only in the CDR-H2 germline substitution (as shown in Table 11B). Compared to P-0734, both P-1175 and P-1181 exhibited substantially reduced potency in blocking PD1 interactions. Notably, P-1174 exhibited significantly reduced potency (EC 50 ) and a 10-fold reduction in E max This corresponds to a 15-fold decrease in potency and a 25% decrease in E max and a 40-50% reduction in fold induction (shown in Figures 7A & 7B and summarized in Table 12). P-1181 showed a more drastic reduction in activity, suggesting that its two distinct CDR germline substitutions, H K62S, H F63V is considered deleterious and therefore the original CDR residue, H Lys62 and H Phe63 will be conserved. The significance of individual CDR residues needs to be evaluated experimentally; these findings suggest that even CDR residues that are close to the interface or not immediately adjacent to antigen contact residues can negatively affect activity.

[0190] Two to five framework residue backmutations were introduced into P-1175 to yield P-1176, P-1177, and P-1178, as detailed in Table 11. As indicated by the data in Figure 8, the backmutations in P-1176, H I69L, H R71T, and H The combination of N73S effectively restored PD1 blocking activity, nearly matching the level of P-0734. Similarly, the activity was not as effective as in P-1176, but the reversion mutation,H V48M and H The combination of S49G and S49G significantly restored the phenotype in P-1177. Nevertheless, the reversion of these two mutations to P-1176 ( H V48M and H Incorporation of the S49G) did not lead to a further increase in activity for the resulting antibody, P-1178 (P-1178 vs. P-1176 in Figure 8 and Table 12). TIFF2025531804000013.tif63170

[0191] P-1198( H N73S), P-1199( H R71T, H N73S), and P-1201( H I69L, H R71T, H By comparing the PD1 blocking activity of the three FR backmutations (N73S), H I69L, H R71T, and H The significance of each of the N73S mutations was further evaluated. As demonstrated in Figure 9, each added back mutation led to a small but significant cumulative increase in PD1 blocking activity. Only the combination of all three back mutations in P-1201 led to a near-complete restoration of functional activity (EC values ​​of 1.28 nM and 0.78 nM for P-1201 and P-0734, respectively). 50 value). Therefore, all three back mutations, H I69L, H R71T, H N73S is considered essential and will be incorporated.

[0192] Furthermore, the PD1 inhibitory activity of P-1194, P-1201, and P-1238 was compared and shown in Figures 10A and 10B. One additional CDR germline substitution, H P-1194 and P-1201, which differ only by F59Y, showed identical PD1 blocking potency when the IGHV1-2 germline sequence was employed. HThis suggests that, contrary to previous observations that the F59Y germline substitution was deleterious, this particular substitution did not negatively affect activity. Therefore, it is conceivable that the impact of individual CDR germline substitutions depends on the context of the surrounding framework sequences. P-1238 was equipotent to the reference antibody, P-0734, with EC values ​​of 0.73 nM and 0.70 nM, respectively. 50 Compared with P-1194, two additional framework reversion mutations in P-1238, H V48M, and H S49G contributed to a small but discernible improvement in activity.

[0193] In the final evaluation, P-1174, P-1193, P-1198, P-1199, and P-1201 were classified as PD1. + The PD1-expressing antibodies P-1174, which fully preserved its PD1-blocking potency (Fig. 6C and 6D), exhibited binding affinity to PD1-expressing cells comparable to that of the reference antibody, P-0734 (Fig. 11A and 11B). P-1198, P-1199, and P-1201, which contain one to three framework backmutations, exhibited subtle but significant differences in potency in blocking PD1 interactions (Fig. 9), but no such variation in activity was detected in cell-based binding assays. All three compounds inhibited PD1. + P-1193 demonstrated equal potency as P-0734 in binding to cells (Figures 11C & 11D and Table 13). However, as shown in Figures 11C & 11D and Table 13, the cell-based binding assay was able to distinguish P-1193, which does not contain framework backmutations, from the other compounds. However, the degree of reduction was less pronounced than that observed in the blocking assay. The data further confirm our previous observation that mechanism-based PD1 / PD-L1 blocking assays are more sensitive than binding assays in identifying subtle activity differences. TIFF2025531804000014.tif58170

[0194] In summary, the optimized PD1-blocking antibodies, P-1194, P-1201, and P-1238, built on a VH framework (IGHV3-23) with substantially lower sequence homology but superior biophysical properties, can fully or nearly fully retain the functional activity of the antibodies and demonstrate improved similarity scores to the closest human germline sequence (IGHV3-23). ​​The mutation details and similarity scores for each antibody are summarized in Table 14. TIFF2025531804000015.tif87170

[0195] Example 6 Germlined substitutions led to reduced hydrophobicity of optimized PD1-blocking antibodies Among 23 FDA- and EMA-approved therapeutic mAbs, pembrolizumab was the most hydrophobic and consequently had the highest tendency to aggregate (Goyon et al., J. Chromatogr. B 1065-1066:35-43, 2017). Consistent with the experimentally determined apparent hydrophobic interaction chromatography (HIC) retention coefficient (k), the SSH2.0 hydrophobicity prediction tool (http: / / i.uestc.edu.cn / SSH2 / ; Zhou et al., Front. Genet. 13:842127, 2022) indicated that both variable chains of pembrolizumab have a significant risk of hydrophobic interactions. The probabilities of hydrophobic interactions for its VH and VL are 0.97 and 0.61, respectively. An antibody is predicted to have a high risk of hydrophobic interactions if the probability is 0.5 or higher (1 being the maximum possible value).

[0196] While the focus of germline substitutions was to enhance the degree of "humanity" in the antibody sequence, the process also resulted in a significant reduction in the probability of hydrophobic interactions for several optimized antibody sequences. Table 15 provides a summary of the predicted probability of hydrophobic interactions for the variable domains of exemplary optimized PD1-blocking antibodies, as estimated by SSH2.0. TIFF2025531804000016.tif87170

[0197] Two light chain CDR germline substitutions, as shown in Table 15: L K27Q and L L54R significantly reduced the hydrophobicity probability of the VL from 0.607 for P-0734 to 0.131. These two amino acid changes were applied to the VL in all optimized PD1-blocking antibodies listed in Table 15. CDR germline substitutions in the heavy chain resulted in only a slight reduction in hydrophobicity, with the hydrophobicity probability changing from 0.971 for (P-0734) to 0.848 for (P-1174) and to about 0.8 for antibodies whose VHs are based on the VH-3 family framework. However, germline substitutions ( H V9A, H T76S, H K82aS, H Q83R, H F84S, H T108L) was implemented into the VH framework of P-1174, the resulting construct, P-1271, had a hydrophobicity probability of 0.185, much lower than that of P-1174.

[0198] Hydrophobic patches on the surface of antibodies are often implicated as one of the main contributors to their tendency to aggregate. Furthermore, these hydrophobic patches can cause high viscosity. Consequently, exemplary PD1-blocking antibodies with significantly reduced hydrophobic potential are expected to exhibit improved biophysical properties. PD1-targeting IL-2 immunocytokines and VitoKine fusions constructed using these optimized PD1-blocking antibodies are also expected to have enhanced developability profiles.

[0199] Example 7 Identification of the optimal IL-2Rα Sushi variant as the cryptic subdomain of IL-2 VitoKine A representative PD1 Ab IL-2 VitoKine construct is shown in Figure 3. Monomeric IL-2 or an IL-2 variant as the active partial domain (D2) is fused between a PD1 antibody (D1) and an IL-2Rα Sushi domain as the cryptic partial domain (D3). Linker 2 (L2) connecting IL-2 and IL-2Rα is protease-cleavable. IL-2 in the VitoKine construct remains inactive until locally activated by proteases present exclusively or upregulated at the tumor site or within the tumor microenvironment (TME). After the L2 linker is cleaved, the cryptic α-subunit ideally dissociates, as shown in Figure 2. Therefore, it is desirable to identify IL-2Rα variants with weakened binding to IL-2 to ensure that the cryptic α-subunit (D3) can readily diffuse after proteolysis but still effectively occlude the activity of the IL-2 partial domain before the linker is cleaved.

[0200] IL-2RαSushi variants were designed to weaken binding to IL-2 by incorporating mutations in residues that interact with IL-2. As listed in Table 16, exemplary IL-2RαSushi variants, P-0751, P-0752, and P-0753, contain Y43A, L42G, and R36A mutations, respectively. They were expressed as monomeric Fc fusion proteins by fusing them to the knob Fc chain of a knob-into-hole heterodimeric Fc chain pair (SEQ ID NOs: 187 and 188). P-0757 is an Fc fusion of monomeric wild-type IL-2RαSushi. The binding ability of these three IL-2RαSushi variants to IL-2 was evaluated using ELISA.

[0201] Briefly, IL-2Rα Sushi variant Fc fusion protein was coated onto the wells of a Nunc Maxisorp 96-well microplate at 1 μg / well. After overnight incubation at 4°C and blocking with 1% BSA, serial dilutions of P-0689, a monomeric wild-type IL-2 equivalent (containing an active neutral C125I mutation; SEQ ID NO: 117) Fc fusion, were added to each well at 100 μL / well. After 1 hour of incubation at room temperature, 100 μL / well of biotin anti-IL-2 antibody clone B33-2 (BD biosciences) was added and incubated for 1 hour at room temperature. Subsequently, 100 μL / well of avidin-HRP (BioLegend) at a dilution of 1:5000 was added. After 60 minutes of incubation and washing, 100 μL / well of TMB substrate (ThermoFisher) was added. Plates were sealed and incubated in the dark at room temperature. The reaction was stopped by adding 2N sulfuric acid (Ricca Chemical). Absorbance was determined at 450 nm and curves were fitted using GraphPad Prism software.

[0202] As summarized in Table 16 and shown in Figure 12, the amino acid substitutions Y43A, L42G, and R36A each affected the interaction with IL-2. The Y43A change resulted in a small reduction (8.1-fold) in IL-2 binding, while the R36A substitution significantly reduced the binding EC 50 This led to a significant 346-fold reduction in binding to IL-2, while the L42G change caused an intermediate, or 35-fold, reduction in binding to IL-2. TIFF2025531804000017.tif52170

[0203] Four Fc IL-2 VitoKine molecules were constructed using the three IL-2RαSushi variants described above along with their wild-type counterparts as the masking domains. Each of these molecules contains a monomeric IL-2 C125I variant (equivalent to wild-type; SEQ ID NO: 117) as the active domain (D2) and a 15-amino acid MMP2 / 9-cleavable L2 linker (SEQ ID NO: 84) connecting IL-2 and IL-2RαSushi (D3). The heterodimeric Fc chain (SEQ ID NOs: 187 and 188) served as the D1 domain. The masking efficiency of these variants was subsequently assessed by assessing their efficacy in inducing Ki67 expression, a marker for cell proliferation, in CD8+ T and NK cells via a human PBMC assay. P-0704, an IL-2 P65R variant (SEQ ID NO: 118) Fc fusion that maintains its wild-type IL-2 potency for the dimeric IL-2Rβγ receptor, was included as a fully active IL-2 control for this set of Fc IL-2 VitoKines.

[0204] Briefly, human PBMCs were isolated by Ficoll-Hypaque centrifugation from buffy coats purchased from the Blood Oklahoma Institute. Purified human PBMCs were treated with serial dilutions of test compounds and incubated at 37°C for 5 days. On day 5, cells were washed once with FACS buffer (1% FBS / PBS) and initially stained with surface marker antibodies, including Fc blocker (BioLegend) and anti-human CD56-FITC and anti-human CD8-APC (BioLegend) at a 1:50 dilution. After 30 minutes of incubation and washing, the cell pellets were thoroughly resuspended in 200 μL / well of 1x Fixation & Permeabilization Standard Solution (Invitrogen) and incubated for 30 minutes at room temperature in the dark. After centrifugation, 200 μL of 1x Permeabilization Buffer (Invitrogen) was added to each well for another wash. The cell pellet was resuspended in permeabilization buffer containing anti-human Ki67-PE (BD Life Sciences) at a dilution of 1:25. After an additional 30 minutes of incubation at room temperature, the cells were collected, washed, and resuspended in FACS buffer and analyzed by flow cytometry. Data were expressed as the percentage of Ki67-positive cells within the gated population. Details of the dose-responsive Ki67 proliferation are shown in Figures 13A and 13B. Additionally, data specific to CD8+ T cells are summarized in Table 17. TIFF2025531804000018.tif62170

[0205] Figure 13 shows that P-0701, which has wild-type IL-2RαSushi as the cryptic partial domain (D3), showed a significant 3-log reduction in the induction of CD8+ T and NK cell proliferation compared to its fully active IL-2 Fc fusion counterpart, P-0704. We hypothesized that the incorporation of IL-2 binding-disrupting mutations into the D3 domain might weaken the cryptic ability of D3, thereby reducing the inactivity of VitoKine. We also speculated that the degree of this reduction would be consistent with the level of reduction in binding strength between IL-2 and the IL-2RαSushi variant.

[0206] As shown in Figure 13A and Table 17, Fc VitoKines P-0754 and P-0756, which contain Y43A and R36A in the IL-2RαSushi D3 domain, exhibited weakened cloning ability compared to P-0701. This led to a higher VitoKine endogenous basal level in stimulating CD8+ T cell proliferation, and the trend was consistent in NK cells, as seen in Figure 13B. The decrease in cloning efficiency, however, did not consistently correlate with the magnitude of the reduction in binding intensity. For example, the Y43A mutation had a minimal effect on binding, exhibiting only an 8.1-fold decrease, while the R36A mutation caused a substantial, approximately 200-fold decrease in binding. Furthermore, despite having 35-fold weaker binding to IL-2, the L42G variant maintained nearly identical cloning efficacy to its wild-type counterpart, as seen in the inactivity of its corresponding VitoKine, P-0755 (Figures 13A and 13B). Although unexpected based on previous knowledge, multiple experiments confirmed that the changes in binding strength resulting from mutations in IL-2RαSushi did not consistently correlate with alterations in its hiding ability, an inconsistency that may be attributed to unique spatial interactions in the VitoKine format.

[0207] Consequently, the IL-2RαSushi L42G variant was selected as the preferred cryptic domain (D3) for IL-2 VitoKine due to its retained cryptic ability to maintain the inactivity of the corresponding VitoKine, given its weakened binding to IL-2, and its potential for easy diffusion upon in vivo proteolysis to achieve full activity. On the other hand, R36A or Y43A can be used as the cryptic domain when tuning the intrinsic basal activity of IL-2 VitoKine to optimize the balance between the desired antitumor efficacy and potential systemic toxicity. Additionally, other IL-2RαSushi variants with varying degrees of reduced binding to IL-2, such as K38E, can be used as the D3 domain of IL-2 VitoKine according to the same rationale.

[0208] Example 8 Amino acid substitutions in p65 resulted in surprisingly diverse effects on binding to IL-2Rα The preferential expansion of regulatory T cells (Tregs) by IL-2 due to the high and constitutive expression of IL-2Rα on Tregs represents an undesirable effect of IL-2 for cancer immunotherapy. IL-2 variants designed to weaken or prevent binding to IL-2Rα reduce their responsiveness to Tregs. IL-2 variants that no longer bind to IL-2Rα are expected to not preferentially activate Tregs, but only at concentrations that also activate CD8+ T and NK cells.

[0209] The P65 residue of the IL-2 molecule is involved in van der Waals interactions with key residues on the IL-2Rα interface, particularly R36 and L42. However, it does not form salt bridges or hydrogen bonds with IL-2Rα (Xinquan Wang, et al., Science (2005), 310:1159-1163). Given this, one might assume that changes to P65 would likely only slightly alter the interaction with the IL-2Rα subunit and cause minimal effects on binding. However, the actual effects of P65 modifications on its interaction with IL-2Rα were surprisingly diverse, ranging from complete maintenance or even improvement of binding to its weakening or complete loss.

[0210] A panel of IL-2 variants with different P65 substitutions was fused to Fc via a flexible GS-based linker (SEQ ID NO: 103) in either dimeric or monomeric formats. All of these variants contain an active neutral C125I mutation, intended to enhance developability. Their binding affinity to IL-2Rα (CD25) was then assessed using ELISA. Briefly, IL-2Rα-ECD was coated onto wells at 0.1 μg / well. After overnight incubation at 4°C and blocking, serial dilutions of IL-2 Fc fusion proteins were added to each well at 100 μL / well. After 1 hour of incubation at room temperature, 100 μL / well of goat anti-human IgG Fc-HRP (diluted 1:5000 in diluent) was added to each well and incubated at room temperature for 1 hour. Plates were developed at room temperature in the dark for 10 minutes after the addition of 100 μL of TMB substrate, followed by the addition of 100 μL / well of stop solution. Absorbance was determined at 450 nm and curves were fitted using Prism software (GraphPad).

[0211] ELISA binding curves are shown in Figure 14. Additionally, ELISA binding EC2 for the IL-2 variants normalized to that of wild type (either P-0531 or P-0689, depending on the valency of each construct) is shown.50 The values ​​are detailed in Table 18. TIFF2025531804000019.tif92170

[0212] As shown in Figures 14A and 14B, the P65G mutation in P-0608, the P65E mutation in P-0633, and the P65A mutation in P-0706 did not appear to impair interaction with the IL-2Rα subunit. Rather, these mutations enhanced binding affinity for IL-2Rα by 18-fold, 10-fold, and 10-fold, respectively, when compared to their respective wild-type IL-2 controls.

[0213] In another set, IL-2 variant Fc fusions, i.e., P-0634, P-0708, and P-0709, had alterations at P65 that led to different levels of impairment in binding to the IL-2Rα subunit. As shown in Figure 14C and detailed in Table 10, the P65N mutation in P-0708 resulted in a moderate 8.6-fold reduction in IL-2Rα binding. On the other hand, the P65H (P-0634) and P65Q (P-0709) alterations had a more pronounced effect, causing a 23-fold and 43-fold reduction in binding, respectively.

[0214] Another group of P65 substitutions, particularly P65R and P65K, appeared to cause profound interference in the IL-2 and IL-R2Rα interaction, completely eliminating binding of P-0635, P-0704, and P-0707 to IL-2Rα, where P-0635 and P-0704 are dimeric and monomeric versions of the IL-2 P65R variant Fc fusion, and P-0707 harbors the P65K amino acid change. Figure 14D reveals that these three IL-2 mutein Fc fusions barely had any detectable binding signal, even at IL-2Rα concentrations as high as 100 nM. This is comparable to a benchmark molecule that has three CD25-interfering mutations, F42A / Y45A / L72G, known to eliminate binding, as reported in Christian Klein et al., OncoImmunology (2017), 6:3, e1277306.

[0215] In summary, changes to the P65 residue led to a diverse range of effects on IL-2Rα binding, including increasing, maintaining, reducing, or completely preventing binding of the resulting IL-2 variants to IL-2Rα. Such a wide range of outcomes, resulting from modifications to what appear to be nonessential amino acids, would not be the predicted outcome of a structure-based mutagenesis approach. The complete loss of IL-2Rα binding was unexpected and not predicted by the prior art. This is particularly surprising given that the P65 mutations only altered a small portion of the van der Waals interaction surface.

[0216] Changes in IL-2Rα binding strength are expected to correlate with IL-2 potency in activating Treg cells. To test this, IL-2 variant Fc fusion proteins with enhanced (P-0608), reduced (P-0634 and P-0709), or abolished (P-0635 and P-0704) binding to IL-2Rα were examined for their ability to differentially stimulate STAT5 phosphorylation in CD4+ Treg cells. STAT5 is known to be involved in the signaling cascade downstream of IL-2 binding to the transmembrane IL-2 receptor. The wild-type IL-2 fusion P-0531 and benchmark molecules were included for comparison.

[0217] Phosphorylation of STAT5 in lymphocyte subpopulations was measured in fresh human PBMCs using the transcription factor FOXP3 to identify Treg populations in FACS analysis. Specifically, serum was removed from purified PBMCs in MACS buffer (Miltenyi Biotech) for 1 hour at 4°C, and subsequently treated with serial dilutions of test compounds for 30 minutes at 37°C. Cells were then fixed, permeabilized, stained with specific antibodies, and further analyzed by flow cytometry following a procedure similar to that detailed in Example 7. Staining was achieved using a mixture of anti-CD25-PE, anti-FOXP3-APC, anti-pSTAT5-FITC, and anti-CD4-PerCP-Cy5.5 antibodies (purchased from BioLegend or BD Life Sciences). Flow cytometry data were analyzed for Treg cell subsets by CD4 / Foxp3 / CD25. high Data are expressed as the percentage of pStat5 positive cells in the gated population.

[0218] As shown in Figure 15, there is a clear correlation between IL-2Rα binding strength and potency in stimulating STAT5 phosphorylation in CD4+ Treg cells. All compounds in Figure 15A feature bivalent IL-2 variants, while all compounds in Figure 15B have monomeric IL-2 variants. P-0608, with enhanced IL-2Rα binding, exhibited appreciably higher potency compared to P-0531. P-0626 (Figure 15A) and P-0709 (Figure 15B) exhibited reduced pSTAT5 potency compared to P-0531 / P-0689, consistent with their reduced IL-2Rα binding strength. However, reduced but residual binding to IL-2Rα ensured that Tregs were activated more effectively than both P-0635 / P-0689 and the benchmark molecule (valency match) that completely lost IL-2Rα binding. Similarly, total loss of IL-2Rα binding led to a significant shift in Treg potency, approximately 5 logs to the right. This residual Treg signaling resulted from activation of IL-Rβγ, which is found on Treg cells.

[0219] Furthermore, all exemplary IL-2 variant Fc fusions containing mutations that enhance, reduce, or abolish binding to IL-2Rα showed unaltered binding to IL-2Rβγ (FIG. 16A). They also showed approximately equal potency in inducing Ki-67 expression in CD8+ T cells (FIG. 16B). The data highlight the fact that IL-2 mutations at the IL-2Rα interface do not alter interactions with IL-2Rβγ, regardless of their effect on IL-2Rα binding.

[0220] Example 9 Identification of IL-2 variants with optimal IL-2Rα binding as active domains for VitoKine The active domain (D2) of the IL-2 VitoKine construct was selected from a panel of IL-2 variants with varying levels of binding strength to IL-2Rα identified in Example 8. Incorporation of IL-2 variants with reduced or eliminated IL-2Rα binding into VitoKine can reduce Treg responsiveness after proteolytic activation in the vicinity of tumors. However, given that the binding between D2 and D3 appears to be necessary for the cloning ability of VitoKine, it is essential to achieve a balance between the degree of weakened IL-2Rα binding and the efficiency of cloning.

[0221] Four exemplary IL-2 VitoKines, namely, P-0800, P-0830, P-0831, and P-0802, all contain the anti-mouse PD1 antibody P-0722 (SEQ ID NOs: 52, 189, and 190) as the D1 domain, the IL-2RαSushi L42G variant (SEQ ID NO: 184) as the hidden partial domain (D3), a non-cleavable linker (SEQ ID NO: 103) as the L1 linker, and an MMP-2 / 9 cleavable linker (SEQ ID NO: 84) as the L2 linker. As detailed in Table 19, the active domain (D2) contains a P65R mutation in P-0800, a P65N mutation in P-0830, and a P65Q mutation in P-0831. P-0802 has an IL-2 wild-type equivalent as the D2 domain.

[0222] Exemplary VitoKines were evaluated for their ability to induce Ki67 expression on CD8+ T cells (FIG. 17A) and NK cells (FIG. 17B) using fresh human PBMCs. P-0782, a non-VitoKine immunocytokine counterpart of P-0800 containing a monomeric IL-2 P65R variant, was included as a fully active IL-2 reference. The EC values ​​of these compounds in terms of stimulation of Ki67 expression in NK cells were 50 The values, along with their fold change compared to P-0782, are summarized in Table 19.

[0223] The data revealed that when the D2 domain was wild-type IL-2, the D3 domain of the VitoKine construct (P-0802) conferred roughly a 3-log reduction in activity, suggesting a strong sequestering ability of the D3 domain. In contrast, for P-0800, which incorporates an IL-2 variant (P65R) with abolished IL-2Rα binding, there was only a 10- to 20-fold reduction in activity, suggesting a significantly weakened sequestering ability of D3 due to the absence of binding between the D2 and D3 domains. Interestingly, when IL-2 variants such as P65N and P65Q, which have intermediately reduced IL-2Rα binding, were used in P-0830 and P-0831, the D3 domain conferred sequestering efficiency comparable to, or only slightly less than, that of wild-type IL-2. TIFF2025531804000020.tif49170

[0224] It was envisioned that the binding affinity threshold between the D2 and D3 domains, combined with the ideal spatial organization of the binding interface, would be pivotal in determining the efficiency of cloning IL-2 VitoKine. Considering that the IL-2 P65Q variant demonstrates significantly reduced binding strength to IL-2Rα (as depicted in Figure 14C and Table 18), yet is still efficiently cloning by IL-2RαSushi L42G and remains inactive as VitoKine, the IL-2 P65Q variant is selected as the preferred D2 domain for IL-2 VitoKine design. The D3 domain is expected to be easily diffused after protease cleavage due to the reduced binding between the D2 and D3 domains. Once biological activity is fully restored by proteolytic activation, this variant is also expected to have a significantly reduced ability to stimulate Treg cells compared to the wild-type variant, as shown in Figure 15B. Nevertheless, other IL-2 variants with reduced IL-2Rα binding, such as P65H and P65N, may also be considered to achieve the right balance between the desired antitumor efficacy and minimizing potential systemic toxicity.

[0225] In addition to the P65 mutation to achieve a balance between weakened binding and effective cloning by IL-2RαSushi, additional mutations that alter the binding affinity of IL-2 to IL-2Rβγ can be incorporated into the D2 domain of VitoKine. These mutations modulate the overall IL-2 response in cells that primarily express the β and γ receptor subunits, such as CD8+ T and NK cells. Through this strategy, the intrinsic basal activity of VitoKine can be fine-tuned, along with its post-proteolytic activation.

[0226] Example 10 IL-2 variants with IL-2Rβγ-blocking substitutions for reduced overall efficacy The selection of mutations that disrupt the IL-2Rβ and common γ chain (γc) was informed by examination of the IL-2 / IL-2R co-crystal structure (PDB code 2B51). Substitution of energy hotspot residues, such as D20 and N88, that directly interact with IL-2Rβ may result in significantly reduced activity and confer poor potency. Consequently, substitutions were introduced at non-essential residues in the IL-2 / IL-2Rβ interface, such as L19. The L19 residue only makes van der Waals interactions with IL-2Rβ, and the resulting mutants are expected to only slightly alter, rather than significantly reduce, the functional activity of IL-2. Furthermore, replacement of L19 with a non-aliphatic residue may contribute to the proposed "vascular toxicity" of IL-2. 19 LDL" motif (Baluna R, Rizo et al., Proc Natl Acad Sci 1999;96:3957-62).

[0227] Exemplary IL-2Rβ-interfering mutations, L19H, L19Q, and L19Y, were introduced into IL-2 in the context of the P65R and C125I mutations in P-0704 to construct a monomeric IL-2 Fc fusion. The P65R mutation resulted in a complete loss of IL-2Rα binding, and the C125I modification was made for developability purposes, and none of these changes affected the functional activity of IL-2 for IL-2Rβγ. The resulting fusion proteins, P-0731, P-0759, and P-0761, were evaluated for efficacy in stimulating Ki67 expression on human CD8+ T cells and NK cells by flow cytometry. The results are depicted in Figures 18A and 18B and detailed in Table 20A. Compared to P-0704, all variants showed a decrease in their ability to promote proliferation in human CD8+ T cells and NK cells. In particular, P-0759 (L19Q) and P-0761 (L19Y) exhibited a small 3-fold decrease in potency, while the L19H mutation in P-0731 led to a more significant 18-25-fold decrease in potency. Potency reduction can also be achieved by incorporating other L19 mutations, such as L19D, L19R, and L19S. TIFF2025531804000021.tif56170

[0228] Similarly, amino acid substitutions at Q126, a residue essential for γc interaction, were made to weaken IL-2 interaction with γc. All these mutations were also introduced into IL-2 in the context of the P65R and C125I mutations. Fc fusions of monomeric IL-2 variants containing the Q126 mutation are listed in Table 20B.

[0229] The increase in Ki67 expression in human CD8+ T cells and NK cells in response to these IL-2 variants compared to P-0704 is depicted in Figures 19A-19F and further summarized in Table 20B. The IL-2 Q126 mutation had varying degrees of impact on CD8+ T and NK cell proliferation. For CD8+ T cells (Figures 19A, 19C, and 19E), minimal 1.5- to 5-fold decreases were observed with Q126N, Q126H, Q126M, Q126F, Q126W, and Q126Y; moderate 5- to 20-fold reductions were observed with Q126R, Q126G, and Q126S; significant 20- to 50-fold decreases were observed for mutations including Q126A, Q126V, Q126E, Q126L, and Q126T; more drastic decreases (>50-fold) were seen with Q126P and Q126I; and Q126D completely abolished activity. A comparable trend in potency change due to Q126 mutations was observed in NK cells (Figures 19B, 19D, and 19F). TIFF2025531804000022.tif139170

[0230] IL-2 potency can be further fine-tuned by combining IL-2Rβ and γc disrupting mutations, as exemplified by P-1247 (IL-2 domain SEQ ID NO: 173) compared with P-1158 and P-0704. In addition to the P65R and C125I mutations in P-0704, P-1158 contains a Q126N mutation, and P-1247 contains an L19Y and Q126N mutation. As shown in Figure 20A, incorporation of the L19Y mutation in P-1247 resulted in an additional 2.6-fold decrease in potency compared with P-1158 (9.2 nM vs. 3.6 nM) and a combined 4-fold reduction in potency compared with P-0704 (9.2 nM vs. 2.3 nM) for stimulating Ki67 expression in human CD8+ T cells. A similar trend was observed in NK cells (Figure 20B). As will be appreciated by those skilled in the art, various combinations of mutations at positions L19 and Q126 can lead to different degrees of activity modulation and are within the spirit and scope of the present invention.

[0231] In summary, in addition to using IL-2Rα-interfering substitutions in IL-2 to limit the unwanted expansion of immunosuppressive Tregs, incorporating IL-2Rβγ-interfering substitutions provides a way to attenuate overall potency for optimal activity. By introducing specific mutations at either L19 or Q126, varying degrees of potency can be achieved. The desired potency of IL-2 can be carefully fine-tuned through a combination of mutations at the L19 and Q126 positions. Reducing potency helps avoid excessive pathway activation and minimize unwanted target sinks. As a result, this strategy can potentially reduce the toxicity associated with IL-2 therapy and improve pharmacokinetics and pharmacodynamics. Incorporation of reduced-potency IL-2 into VitoKine helps fine-tune its post-proteolytic activation in addition to its intrinsic basal activity.

[0232] Example 11 Construction of PD1 Ab-IL-2 VitoKine using optimized PD1 blocking antibodies and preferred IL-2 and IL-2Rα Sushi domains Antibodies that block PD1 and thus avoid its immunosuppressive effects in the tumor microenvironment may enhance IL-2 responses and further enhance immunity against tumors. The PD1 antibodies used to construct PD1 Ab-IL-2 VitoKine as the D1 domain were selected from optimized human PD1-blocking antibodies containing the light chain sequence set forth in SEQ ID NO: 44 and the heavy chain sequences set forth in SEQ ID NOs: 45-49. These optimized PD1-blocking antibodies have high affinity for the human PD1 protein and demonstrate equal or comparable efficacy to pembrolizumab in blocking PD1. They also have higher sequence similarity scores to their closest human germline sequence, resulting in an improved degree of humanity compared to pembrolizumab. Furthermore, they are predicted to have lower hydrophobicity, which in turn may reduce aggregation tendency compared to pembrolizumab. PD1-targeted IL-2 VitoKine constructed using these optimized PD1-blocking antibodies is also predicted to have an enhanced developability profile.

[0233] Table 21 lists exemplary PD1 Ab-IL-2 VitoKines, whose structures are depicted in Figure 3A. All exemplary VitoKines contain the IL-2 P65Q variant with or without a mutation for modulating activity toward IL-2Rβγ as the active partial domain (D2), the IL-2RαSushi L42G variant as the cryptic partial domain (D3), and a cleavable L2 linker (SEQ ID NO: 84) connecting the D2 and D3 domains. Nevertheless, if it is desired to adjust the intrinsic basal activity of the IL-2 VitoKine, other IL-2RαSushi variants, such as R36A, can be used as the cryptic partial domain. Additionally, the L1 linker connecting the PD1 Ab and IL-2 can also be cleavable. The composition of the cleavable linker can be further optimized by using various sequences set forth in SEQ ID NOs: 78-94 to better suit different disease conditions and / or stages. TIFF2025531804000023.tif92170

[0234] All genes were codon-optimized for expression in mammalian cells, synthesized, and subcloned into recipient mammalian expression vectors via the services of GenScript. VitoKine constructs were produced by co-transfecting the mammalian expression vectors into Expi293 cells (ThermoFisher) according to the manufacturer's instructions. Protein purification and characterization were performed according to the same procedures detailed in Example 2.

[0235] PD1 Ab-IL-2 VitoKine constructs containing optimized antibody sequences, including P-1197, P-1239, and P-1272, were found to be expressed at substantially higher levels than P-1120, which contains the reference antibody, P-0734. Under identical transient expression conditions using the same batch of Expi293 cells, P-1197, P-1239, and P-1272 were expressed at titers of 137–150 mg / L compared to 60 mg / L for P-1120. These data suggest that PD1-blocking antibodies with optimized sequences that eliminate potential sequence constraints may lead to improved developability of the corresponding VitoKine constructs.

[0236] Because these optimized PD1 antibodies did not react with mouse PD1, surrogate mouse PD1-Ab-IL-2 VitoKines and additional controls were similarly prepared. These were used for in vivo studies, particularly for pharmacokinetic (PK) / pharmacodynamic (PD) and tumor experiments in immunocompetent mice. Table 21B provides detailed information about these molecules. All VitoKine constructs listed in this table incorporate the same D3 domain (SEQ ID NO: 184). With the exception of P-871, these VitoKines contain the mouse PD1 antibody P-0722 (SEQ ID NOs: 189, 190, and 52) as the D1 domain. P-0871, the non-targeting VitoKine counterpart of P-0831, contains the germline antibody P-1260 (SEQ ID NOs: 192, 193, and 194) as the D1 domain. P-0877 is a non-cleavable VitoKine, and P-0838, which lacks the L2 linker and D3 and whose structure is shown in Figure 3B, serves as the non-VitoKine immunocytokine counterpart to P-0831. TIFF2025531804000024.tif88170

[0237] While P-0831 is the primary subject for the in vivo studies in the following examples, other VitoKines containing IL-2 variants with additional mutations to target IL-2Rβγ (e.g., those listed in Table 21B) are expected to achieve similar antitumor efficacy when dosages are adjusted appropriately. Because the intrinsic basal activity of IL-2 VitoKines is directly correlated with the activity of their active domain (D2), attenuated D2 activity and proportionally modulated VitoKine basal activity allow for the administration of higher doses without adverse effects. This fully supports the function of PD1 antibodies to reverse T cell anergy or exhaustion, thereby potentially enhancing synergy with IL-2 immunotherapy and helping to expand the therapeutic window.

[0238] Example 12 Ex vivo activity and in vitro proteolytic activation of PD1 Ab-IL-2 VitoKine It is important that PD1 antibodies retain their binding and functional activity when incorporated into PD1 Ab-IL-2 VitoKine. PD1 antibodies with excellent target binding and PD1 blocking function can enhance the specificity and selectivity of TIL targeting and further cooperate with IL-2 anti-cancer immune responses by effectively reversing T cell anergy and exhaustion.

[0239] In a comparative analysis using a luciferase reporter assay, the PD1 inhibitory potency of exemplary VitoKine P-1197, P-1239, and P-1272 was set against their respective PD1 blocking antibodies, P-1174, P-1238, and P-1271. As shown in Figures 21A and 21B, each of the three antibodies not only maintained but slightly improved their blocking potency when incorporated into their corresponding VitoKine constructs. For example, the PD1 antibodies P-1174, P-1238, and P-1271 had EC values ​​of 1.29 nM, 1.82 nM, and 1.53 nM, respectively. 50 On the other hand, their counterparts VitoKine, P-1197, P-1239, and P-1272 blocked the PD1 / PD-L1 interaction with EC values ​​of 0.92 nM, 1.27 nM, and 1.20 nM, respectively. 50 Additionally, in the VitoKine format, E max There was an 11-17% increase in both phenotype and fold induction.

[0240] Figure 22 further confirms that, regardless of the specific PD1 antibody composition, IL-2 activity remains effectively cloaked by the IL-2Rα Sushi domain. The exemplary PD1 Abs IL-2 VitoKine, P-1197, P-1272, and P-0872, which differ only based on their distinct PD1 blocking antibodies (detailed in Table 21A), all show a ∼300-fold decrease in their ability to induce CD8+ T and NK cell proliferation when compared to P-0879 (Figures 22A and 22B) or P-1273 (Figures 22C and 22D). For context, P-0879 and P-1273 are the non-VitoKine immunocytokine counterparts of P-1272, respectively, lacking the cryptic D3 domain. EC 50 Values ​​can be found in Table 22. For CD8+ T cells, the low potency of VitoKine prevented curve fitting, resulting in only approximate EC 50 Values ​​were obtained as results. P-1174 is the PD1 antibody component of P-1197 and was included as a negative control. TIFF2025531804000025.tif55170

[0241] P-1272 was further evaluated for in vitro proteolytic activation. Consistent with other exemplary PD1 Ab-IL-2 VitoKine in this invention, P1272 contains a single MMP-2 / 9 cleavable L2 linker (SEQ ID NO: 84). Procedurally, 3.3 μg of latent MMP-2 (BioLegend) was first activated with APMA (Millipore Sigma) according to the manufacturer's instructions, then buffer-exchanged and added to 120 μg of P-1272 in 0.4 ml of the manufacturer's recommended assay buffer (100 mM Tris, 20 mM CaCl, 300 mM NaCl, 0.1% (w / v) Brij 35, pH 7.5). After a 3-hour incubation at 37°C, the processed sample was then purified using Protein A resin (MabSelect SuRe; Cytiva) in bind-elute mode. The eluted samples were analyzed in reducing SDS-PAGE gels and their biological functions were assessed in ex vivo functional assays.

[0242] Figure 23A shows that the cryptic moiety of P-1272 was efficiently and completely cleaved, resulting in P-1272-Activ., which corresponds to the active form 2 depicted in Figure 2. Efficient in vitro proteolysis led to full restoration of IL-2 activity, exemplified by the indistinguishable activity of P-1272-Activ. and P-1273 in inducing dose-dependent expression of Ki67 in CD8+ T cells from fresh human PBMCs (Figure 23B). Comparable findings were observed with other PD1 Ab-IL-2 VitoKines, as exemplified by P-0831, depicted in Figure 23C.

[0243] Furthermore, a distinct form of PD1 Ab-IL-2 VitoKine, P-1345, differs from other VitoKines in that it contains a cleavable L1 linker (SEQ ID NO: 84) and a non-cleavable L2 linker (SEQ ID NO: 115). P-1345 was similarly activated through in vitro protease cleavage and its single activated form, referred to as Active Form 1 in Figure 2, was isolated. This form was then evaluated for its potency in inducing Ki67 expression in CD8+ T cells of human PBMCs. As demonstrated in Figure 23D, proteolytic activation led to a ∼50-fold increase in activity compared to its intact VitoKine form. However, activation did not fully restore the activity of the IL-2 domain, showing a 6-fold lower activity than its non-VitoKine counterpart, P-0838, and the respective EC 50 The values ​​were 8.29 nM and 1.34 nM.

[0244] These results suggest that using a cleavable L2 linker is advantageous over a cleavable L1 linker, because the active form 2 resulting from cleavage of the L2 linker is a fully functional IL-2 domain fused to the PD1 Ab. This form can activate IL-2R signaling in PD1-expressing T cells near the disease site, enhancing both pathways and synergizing anti-cancer immune responses while reducing systemic toxicity. On the other hand, active form 1 exhibits reduced potency, a shorter half-life, and lacks TIL targeting capability.

[0245] These observations also suggest that the cryptic domain alone is insufficient for effective occlusion of the active domain, resulting in a occlusion efficiency that is approximately 6-fold lower. For efficient occlusion of IL-2 activity, the structure must be in the form of the VitoKine platform, disclosed herein and elsewhere by the inventors in WO2019246392 and WO2021119516, which involves coupling of both a targeting domain (D1) and a occlusion domain (D3).

[0246] Example 13 Prolonged in vivo half-life of PD1-Ab-IL-2 VitoKine in non-tumor-bearing mice The IL-2 domain of VitoKine is designed to remain inactive until locally activated by proteases upregulated in diseased tissue. As a result, binding of IL-2 VitoKine to IL-2 receptors on cell surfaces in peripheral and non-diseased tissues is expected to be significantly reduced. This mitigates potential antigen sink and / or target-mediated deposition, resulting in an extended in vivo half-life. A study was performed to compare the pharmacokinetics of the murine PD1 Ab IL-2 VitoKine, P-0831, with its non-VitoKine immunocytokine counterpart, P-0838, in non-tumor-bearing C57BL / 6 mice.

[0247] Seven-week-old naive female C57BL / 6 mice were received from Charles River Laboratory. Mice were acclimated in-house for 7 days before the start of the study. At the start, P-0831 and P-0838 were administered intravenously at a dose of 1 mg / kg each. Vehicle (PBS) was included as a negative control. Blood samples were collected by buccal bleed at 10 minutes, 2 hours, 6 hours, 24 hours, 48 ​​hours, 72 hours, 120 hours, 168 hours, 240 hours, and 360 hours after injection. Each group consisted of three mice, and blood was collected either weekly or every three days, with a maximum frequency of two times per group.

[0248] Serum concentrations of the compounds were determined using ELISA assays. Three different ELISA methods were developed for P-0831 to measure: 1) total VitoKine concentration (including both activated and intact forms); 2) intact VitoKine concentration; and 3) activated VitoKine concentration. For all three methods, maxisorp plates were coated with mouse PD1 protein (R&D systems) overnight at 4°C. After this, the plates were blocked with Superblock (ThermoFisher). Blood samples at various dilutions were added to the plates and incubated for 1 hour at room temperature.

[0249] For total VitoKine detection, an anti-IL-2 goat polyclonal antibody (R&D Systems) was added, followed by a secondary HRP-conjugated donkey anti-goat IgG (ThermoFisher). To detect intact VitoKine, a polyclonal anti-CD25 antibody (R&D Systems) was used, which was probed with HRP-conjugated streptavidin protein (ThermoFisher). For activated VitoKine detection, a biotinylated monoclonal anti-IL-2 antibody (BD Pharmingen) paired with HRP-conjugated streptavidin was applied. For P-0838 detection, the same anti-IL-2 goat polyclonal antibody (R&D Systems) used to detect total VitoKine concentration was used, followed by donkey anti-goat IgG-HRP. The resulting signal was developed using Ultra TMB substrate solution, and values ​​were extrapolated from a nonlinear regression curve fit in GraphPad Prism.

[0250] As shown in Figure 24, the concentration profiles for intact P-0831 and total P-0831 (including both intact and activated forms) closely matched, suggesting that P-0831 circulates predominantly in its intact state. Furthermore, there was no evidence of activated P-0831 detected at any time point after administration, confirming the notion that P-0831 remains intact in the periphery.

[0251] In contrast to the concentration profile of P-0831, which remained measurable 360 ​​hours after a 1 mg / kg dose, the serum concentration of P-0838 declined rapidly. It was substantially lower by 72 hours after dosing and undetectable by 120 hours. The gray dashed horizontal line in Figure 24 represents the lower limit of quantitation (LLOQ) for serum P-0838 levels. For measurements that fall below the LLOQ, values ​​are calculated as 10 -3 Assigned as nM.

[0252] The findings strongly support the concept that the VitoKine format is superior in extending the in vivo half-life of the active domain. The significantly prolonged in vivo half-life of VitoKine is believed to result from the inactivity of the IL-2 domain in peripheral blood. This inactivity likely reduces interaction with IL-2 receptors on cell surfaces in both peripheral and non-diseased tissues, significantly reducing cell activation and proliferation, and consequently mitigating potential antigen sink and / or target-mediated deposition.

[0253] Example 14 Minimized systemic pharmacodynamic effects of PD1-Ab-IL-2 VitoKine in non-tumor-bearing mice The VitoKine platform aims to mitigate systemic on-target toxicity and expand the therapeutic window for cytokine therapy. This is achieved by rendering the active cytokine inactive within the construct, preventing it from interacting with receptors in peripheral blood or on the surface of non-diseased cells. This design helps limit overactivation of cytokine pathways and reduces the risk of unwanted "on-target" effects in "off-tissue" locations. To evaluate this hypothesis, we administered the murine PD1 Ab-IL-2 VitoKine P-0831 to non-tumor-bearing C57BL / 6 mice and assessed its systemic effects compared to its non-VitoKine immunocytokine counterpart, P-0838, by monitoring the proliferation and expansion of peripheral blood lymphocytes over a given period of time.

[0254] Seven- to nine-week-old naive C57BL / 6 mice (n = 4 / group) were administered a single intraperitoneal injection of P-0831 at 2 and 10 mg / kg and P-0838 at 0.3, 1, and 2 mg / kg. Vehicle (PBS) was included as a negative control. Blood samples were collected in heparinized tubes for immunophenotyping analysis on days 0, 3, 5, 7, and 10 post-dosing.

[0255] Subsequently, heparinized blood samples were stained with a panel of antibodies targeting common surface immune cell markers. After lysis of red blood cells using BD Pharmingen lysis buffer, the total number of viable mononuclear blood cells was determined by excluding dead cells with trypan blue. Lysed immune cells were then fixed and permeabilized for 30 minutes in the dark at room temperature using fixation / permeabilization buffer (eBioscience). After washing, these cells were intracellularly stained with an antibody for the Ki67 proliferation marker. Distinct immune cell subsets were identified, and their absolute counts in the circulation were quantified using a flow cytometer (Beckton Dickinson). This was performed using commercially available antibodies: CD3-APC.Cy7, CD8-Percp-cy5.5, CD335-APC, CD45-AF700, CD4-BV421, CD25-BV510, Foxp3-FITC, Ki67-PE, and Granzyme B-BV421. Flow cytometry analysis was performed using FlowJo software and results were plotted using GraphPad Prism.

[0256] In Figure 25, the data revealed that P-0838 dramatically expanded peripheral blood CD8+ T cells (Figure 25A) and granzyme B+ CD8+ T cells (Figure 25B) at doses of 1 and 2 mg / kg, demonstrating a dose-dependent response. Notably, at a dose of 2 mg / kg for P-0838, CD8+ T cells expanded from a baseline level of 900 cells / μL to 2400 cells / μL (a 2.7-fold increase) on day 3. Expansion peaked at 4200 cells / μL (a 4.7-fold increase) on day 5 before declining to near baseline levels on day 7. At a dose of 1 mg / kg for P-0838, peak expansion for CD8+ T cell expansion was observed on day 3 with a 2.3-fold increase and returned to near baseline levels by day 7. At the lower dose of 0.3 mg / kg of P-0838, CD8+ T cell expansion was only slightly detectable. A similar trend was observed in the expansion of cytotoxic granzyme B+ CD8+ T cells (Figure 25B). In sharp contrast, even at higher doses of 2 mg / kg and 10 mg / kg, VitoKine P-0831 did not significantly expand CD8+ T cells or granzyme B+ cells throughout the 10-day period (shown in Figures 25A and 25B).

[0257] Consistent with the results of the ex vivo assay, NK cells showed a higher responsiveness to IL-2 treatment than CD8+ T cells. This was evident from the significant NK cell expansion observed at a dose of 0.3 mg / kg of P-0838 (Figure 25C). NK cell expansion showed dose-dependence between the 0.3 mg / kg and 1 mg / kg doses, with no significant difference between the 1 mg / kg and 2 mg / kg doses. The peak expansion for NK cells among all three doses of P-0838 was on day 3 (Figure 25C). A similar pattern was observed for cytotoxic granzyme B. +This was observed in the expansion of NK cells (Figure 25D). In contrast, P-0831 treatment, even when administered at a significantly higher dose of 10 mg / kg, led to only a slight and delayed increase in the numbers of both NK cells and Granzyme B+ NK cells (Figures 25C and 25D).

[0258] In summary, when compared to the active IL-2 fusion molecule P-0838, P-0831 exhibited significantly reduced systemic proliferation and expansion of specific lymphocytes, highlighting the effectiveness of the VitoKine format in concealing IL-2 activity, thereby preventing unwanted activation of the IL-2 pathway and mitigating the risk of unwanted "on-target" effects in "off-tissues."

[0259] Example 15 Alleviation of cytokine-associated toxicity in mice using PD1 Ab-IL-2 VitoKine Cytokine-related toxicity, also known as cytokine release syndrome (CRS), is one of the major risks associated with cancer immunotherapy. CRS results from an intense immune response and is often associated with elevated circulating levels of several cytokines, including interleukin-6 and interferon gamma (INFγ). As the efficacy of immune-based therapies increases, the magnitude of immune activation may potentially escalate CRS to life-threatening levels beyond those occurring in more natural settings. Considering that the VitoKine platform is designed to limit cytokine pathway overactivation, and considering the proven ability of the exemplary IL-2 VitoKine P-0831 to minimize the systemic activation and expansion of targeted lymphocyte populations (see Example 14), VitoKine holds the promise of significantly reducing cytokine-related toxicity.

[0260] To investigate the potential for reducing cytokine-associated toxicity, we administered varying doses of the murine PD1 Ab-IL-2 VitoKine P-0831 and its non-VitoKine immunocytokine counterpart P-0838 to non-tumor-bearing naive C57BL / 6 mice. Subsequently, we determined circulating levels of INFγ, a key serum proinflammatory cytokine.

[0261] Naive C57BL / 6 mice, 7-9 weeks old and grouped in sets of three (n=3), received a single intraperitoneal injection of P-0831 at doses of 1, 3, 6, 10, and 20 mg / kg and P-0838 at doses of 1, 3, and 6 mg / kg. Vehicle (PBS) and the murine PD1 antibody P-0722 (containing a homodimeric Fc with SEQ ID NOs: 52 and 53) were included as negative controls. Serum samples were collected and isolated from mice 48 hours after treatment. Serum IFNγ concentrations were determined using a mouse IFNγ DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions.

[0262] Figure 26A and accompanying Table 23 reveal that treatment with both P-0831 and P-0838 resulted in a dose-dependent increase in serum IFNγ levels. However, VitoKine P-0831 exhibited significantly reduced IFNγ serum levels compared to P-0838. At a dose of 1 mg / kg, P-0838 treatment resulted in an IFNγ concentration of 253 pg / mL, while P-0831 resulted in only 10.6 pg / mL. More strikingly, at a dose of 3 mg / kg, serum IFNγ for P-0838 rose to 12,482 pg / mL, a 50-fold increase over that seen at the 1 mg / kg dose. In contrast, P-0831, when dosed at 3 mg / kg, demonstrated only a 2.5-fold increase in IFNγ levels compared to its 1 mg / kg dose. Surprisingly, even when administered at a dose of 20 mg / kg, P-0831 only led to small INFγ levels of 182 pg / mL, still below the levels seen with the 1 mg / kg dose of P-0838. As expected, neither vehicle nor antibody treatment induced any appreciable release of this proinflammatory cytokine, as shown in Figure 26A. TIFF2025531804000026.tif72170

[0263] The strong increase in circulating INFγ levels observed in the 3 and 6 mg / kg P-0838-treated groups, resulting from high levels of systemic immune activation, may have led to severe toxicity. In a parallel experiment, naive C57BL / 6 mice (7–9 weeks old, n = 4 / group) were administered P-0831 at doses of 3, 6, and 20 mg / kg and P-0838 at doses of 1, 3, and 6 mg / kg via intraperitoneal injection. Such cytokine-related toxic effects were accompanied by significant weight loss (Figure 26B) and other signs of stress in mice treated with 3 mg / kg and 6 mg / kg P-0838. Given that the established protocol requires the termination of any mouse experiencing more than 10% of its body weight, all mice in the P-0838 6 mg / kg treatment group did not survive beyond 4 days, and three of the four mice in the 3 mg / kg P-0838 group had to be sacrificed on day 4 after a single injection. In contrast, mice treated with P-0838 at all tested doses, even up to 20 mg / kg, remained alive after three doses on a Q2W dosing schedule and did not exhibit significant weight loss (Figure 26B). These suggested that the VitoKine platform exhibited a significantly lower toxicity profile.

[0264] In summary, PD1 Ab IL-2 VitoKine markedly mitigated cytokine-associated toxicity in mice, as evidenced by markedly reduced circulating levels of inflammatory cytokines, exemplified by INFγ, and minimal changes in body weight even at much higher doses compared to non-VitoKine immunocytokine counterparts. The PD1 Ab-IL-2 VitoKine platform effectively minimizes off-target toxicity, thereby providing a wider therapeutic window.

[0265] Furthermore, the ability of PD1 Ab IL-2 VitoKine to be tolerated at much elevated doses provides greater flexibility in optimizing dosing regimens. At higher doses, the ability of PD1 antibodies to reverse T cell anergy or exhaustion is entirely feasible because the dose level is within its effective range, potentially enhancing synergy with IL-2 immunotherapy. Additionally, the incorporation of IL-2 with reduced potency, achieved by introducing mutations that disrupt IL-2Rβ or γc interactions, could lead to VitoKine with lower intrinsic basal activity and potentially further expand the therapeutic window.

[0266] Example 16 Inhibition of established tumor growth in mice by PD1 Ab-IL-2 VitoKine The antitumor efficacy of PD1 Ab IL-2 VitoKine P-0831 was investigated in comparison with its non-VitoKine immunocytokine counterpart P-0838 in a syngeneic MC38 mouse colon cancer model. In these experiments, 7-9 week old female C57BL / 6 mice were injected with 5 × 10 subcutaneously in the right flank. 5 MC38 colon cancer cells were implanted. Approximately two weeks later, tumors grew to approximately 75 mm 3 Once tumors had grown to an average volume of 1000 mg / kg, mice were randomized into groups of 8 on study day 0. On study day 1, the following treatments were given: murine PD1 antibody P-0722 at 9 mg / kg, P-0831 at varying dosage levels (3 mg / kg, 6 mg / kg, and 9 mg / kg), and P-0838 at 1 mg / kg. These treatments were given intraperitoneally every 10 days (Q10D) for a total of two doses. Vehicle (PBS) was used as a control. Both tumor growth and mouse weight were monitored twice weekly. Caliper measurements were used, and volume = 0.5 x (width) 2 Tumor volume (TV) was determined by calculating the tumor growth inhibition (TGI, %) as follows: TGI (%) = [1 - (TV of treated group) / (TV of control group)] x 100 (%). Based on established criteria, tumors were measured for tumors exceeding 1500 mm 3Mice were euthanized when the tumor grew to or exceeded 100 mg / kg / day or became necrotic.

[0267] Figures 27A-27D show tumor growth curves for individual mice for the four different treatment groups containing the IL-2 moiety. Each line in the graph represents one mouse, with the mean tumor growth of the vehicle group represented by a dotted line. Small arrows below the x-axis indicate each dose. Treatment with 6 mg / kg P-0831 demonstrated the most significant and sustained effect, with all eight mice from this group completely eradicating tumor growth by day 45, 34 days after the second and final treatment (Figure 27B). Similarly, in the group treated with 9 mg / kg P-0831, seven of eight mice remained tumor-free at the end of the study (Figure 27C). On the other hand, P-0831 administered at 3 mg / kg had slightly lower efficacy, with five of eight mice remaining tumor-free, although three mice showed tumor growth after an initial period of delayed tumor growth (Figure 27A). For mice treated with 1 mg / kg P-0838, 6 of 8 mice remained tumor-free by the end of the study (FIG. 27D).

[0268] The mean tumor volume, along with the standard error of the mean (SEM) for each group as a function of time, is further shown in Figure 27E. Mice treated with vehicle rapidly developed large subcutaneous tumors. PD1 antibody treatment showed limited efficacy, resulting in a 27% tumor growth inhibition (TGI) compared to the vehicle group. Conversely, all other treatment groups exhibited high efficacy in inhibiting tumor growth, with a TGI of 100% at 45 days after the start of treatment.

[0269] Figure 27F shows that P-0831 was well tolerated with little or no weight loss, even at significantly higher doses than P-0838. It was previously shown (as shown in Figure 26B) that P-0838 was not tolerated at doses of 3 mg / kg or higher. The combined in vivo findings indicate that a 6 mg / kg dose of P-0831 led to a more pronounced and prolonged response. Remarkably, this antitumor efficacy was achieved with significantly lower peripheral lymphocyte proliferation and expansion (as seen in Figure 25), as well as significantly reduced production of circulating INF-γ (Figure 26) when compared to the effects of a 1 mg / kg dose of P-0838. This efficacy is partially attributed to the high-dose tolerability afforded by the VitoKine® format. Consequently, the PD1 Ab IL-2 VitoKine platform offers a broader therapeutic window, allowing the antibody component to achieve its full potential in reversing T cell anergy and exhaustion.

[0270] In a parallel study, immunohistochemistry (IHC) was used to evaluate the impact of PD1 Ab-IL-2 VitoKine on tumor tissue obtained 5 days after treatment. In this preparation, MC38 subcutaneous tumors were similarly established. After randomization (5 mice / group), mice were treated with a single dose of vehicle, P-0722 (6 mg / kg), P-0831 (6 mg / kg), or P-0838 (1 mg / kg). Five days after treatment, mice were euthanized, tumors were extracted, and tissue samples were prepared. Tissue sections were fixed in 10% formalin, paraffin-embedded, processed, and stained with antibodies using HistoWiz according to the manufacturer's instructions to evaluate immune cells in the tumor tissue. Representative IHC images for each group are shown in Figure 28.

[0271] Figure 28 shows that P-0831 treatment induced massive infiltration of CD3+ and CD8+ T cells into tumor tissue. Furthermore, the infiltrated CD8+ T cells were characterized by strong cytotoxic activity, evidenced by strong granzyme B expression. These observations further corroborated the antitumor efficacy data, highlighting the expansion of the number and activity of cytotoxic CD8+ T cells in the tumors of P-0831-treated mice. In sharp contrast, 6 mg / kg of the murine PD1 antibody RO0722 induced only minimal tumor-infiltrating lymphocyte (TIL) presence. P-0838 treatment led to limited T cell infiltration, which demonstrated high granzyme B expression. Importantly, neither treatment resulted in a significant increase in the presence of inhibitory FOXP3+ cells.

[0272] Taken together, PD1 Ab IL-2 VitoKine, exemplified by the surrogate molecule P-0831, effectively inhibited tumor growth by promoting massive infiltration of cytotoxic T cells into tumor tissue while minimizing peripheral lymphocyte proliferation and expansion. Consequently, the use of the VitoKine format may mitigate challenges commonly associated with fully active cytokines, such as excessive stimulation of immune pathways, undesirable on-target and off-tissue toxicity, and unwanted target sinks, while still demonstrating robust antitumor efficacy. Importantly, the compatibility of the PD1 Ab-IL-2 VitoKine with higher dosages ensures that the antibody arm can optimally target and reverse T cell anergy and exhaustion, enhancing existing immune responses. This results in further enhanced immune system activity against tumors. Additionally, the incorporation of IL-2 with reduced potency, achieved by introducing mutations that disrupt IL-2Rβ or γc interactions, results in VitoKine with lower intrinsic basal activity and activity after activation, which could potentially further expand the therapeutic window.

[0273] Example 17 In vivo activity of PD1 Ab-IL-2 VitoKine depends on proteolytic activation and relies on PD1 targeting Using a murine CT26 colon cancer tumor model, we investigated the essential role of VitoKine activation in antitumor efficacy by comparing P-0831 and its non-cleavable VitoKine counterpart, P-0877. The only difference between P-0831 and P-0877 is the L2 linker connecting the IL-2 (D2) and IL-2Rα (D3) domains (see Table 21B for details). As depicted in Figures 29A and 29B, P-0877 showed identical activity to P-0831 in inducing Ki67 expression in CD8+ T and NK cells in human PBMCs, but the IL-2 domain in P-0877 remains hidden and inactive because the D3 domain cannot be cleaved and lead to activation.

[0274] For this study, 7- to 9-week-old female Balb / C mice were treated with 5 × 10 subcutaneous injections into the right flank. 5 CT26 cells were injected. On day 11, the average tumor volume was approximately 75 mm 3 When tumor size reached 1500 mm, mice were randomized into five groups containing eight animals each. Mice received two intraperitoneal injections of 10 mg / kg vehicle (PBS) or P-0722, P-0831, or P-0877 Q12D starting on Study Day 1, the day after randomization. Tumor size and body weight were monitored twice weekly. Based on established criteria, tumors were assessed for size and mass when tumors reached 1500 mm. 3 Mice were euthanized when the tumor grew to or exceeded 100 mg / kg / day or became necrotic.

[0275] The CT26 syngeneic tumor model is generally less responsive to PD1 treatment than the MC38 model. As depicted in Figure 29C, tumors eventually developed in all mice. Treatment with the murine PD1 antibody P-0722 resulted in only a slight delay in tumor growth, resulting in a 25% tumor growth inhibition (TGI). On the other hand, administration of P-0831 at the same dosage demonstrated significantly improved efficacy, exhibiting an 81% TGI. In sharp contrast, P-0877, which possesses a nonactivatable, inactive IL-2 domain, did not demonstrate any improvement in tumor growth inhibition compared to P-0722. These findings suggest that the enhanced antitumor efficacy of VitoKine molecules depends on enzymatic cleavage of the linker to release the cryptic moiety, thereby activating the IL-2 domain around the tumor. It is noteworthy that all tested compounds were well tolerated in mice when given at 10 mg / kg, with no evidence of weight loss, as shown in Figure 29D.

[0276] In a parallel study using the CT26 tumor model, the importance of PD1 targeting for the antitumor efficacy of IL-2 VitoKine was evaluated by comparing P-0871, a non-targeting IL-2 VitoKine, with P-0831. Both P-0871 and P-0831 share the same D2 and D3 domains as well as the L1 and L2 linker, but the D1 domain of P-0871 is the same as that of the non-targeting germline antibody P-1260, which has heterodimeric heavy and light chains with SEQ ID NOs: 191, 192, and 193.

[0277] Mice bearing subcutaneously implanted CT26 tumors were administered two intraperitoneal injections of either vehicle (PBS), P-0722, P-0831, or P-0871 at a dosage of 10 mg / kg Q12D. Figure 30 depicts the mean tumor volume (with SEM) for each group as a function of time. The results show that P-0831 was significantly more effective in delaying tumor growth compared to its non-targeted counterpart, P-0871. This suggests that targeting PD1 is crucial in enhancing the antitumor efficacy of P-0831.

[0278] In conclusion, our findings strongly suggest that the antitumor effect of PD1 Ab-IL-2 VitoKine depends on an in vivo proteolytic cleavage process that subsequently leads to IL-2 activation. Furthermore, the efficacy of this VitoKine appears to be closely linked to its ability to target PD1, indicating that PD1 targeting plays an important role in its therapeutic potential.

[0279] Example 18 Construction and ex vivo characterization of PD1 Ab-IL-2 immunocytokine Linking IL-2 variants to PD1 antibodies aims to deliver IL-2 variants preferentially in cis to PD1+ cells, such as activated and exhausted CD8+ T cells in the tumor microenvironment, to promote selective signaling. This strategy can also reduce systemic IL-2 exposure and provide synergy by removing negative regulation and revitalizing T cells in both function and numbers. In addition to the VitoKine platform, using IL-2 variants with reduced / eliminated binding to IL-2Rα and attenuated IL-2Rβγ activity offers an alternative approach to balancing the ratio between cytokine and antibody arms to exhibit dramatically different potencies and molecular weights than the native version. This balance allows for optimal dosing and preserves the function of each arm. Reduced cytokine activity is expected to minimize peripheral activation, mitigate in vivo antigen sink and target-mediated deposition, and facilitate tumor targeting via the antibody arm.

[0280] The PD1 antibodies used to construct the PD1 Ab-IL-2 immunocytokine were selected from optimized human PD1 blocking antibodies containing the light chain sequence set forth in SEQ ID NO: 44 and the heavy chain sequences set forth in SEQ ID NOs: 45-49. These optimized PD1 blocking antibodies have high affinity for the human PD1 protein and demonstrate equal or comparable potency to pembrolizumab in blocking PD1. They also have higher sequence similarity scores to their closest human germline sequence, resulting in an improved degree of humanity compared to pembrolizumab. Furthermore, they are predicted to have lower hydrophobicity, which in turn is likely to reduce aggregation tendency compared to pembrolizumab. PD1-targeting IL-2 immunocytokines constructed using these optimized PD1 blocking antibodies are also predicted to have an enhanced developability profile.

[0281] To create a PD1 Ab monomeric IL-2 immunocytokine fusion, the IL-2 variant is fused to the C-terminus of the knob-containing heterodimer heavy chain of the PD1 antibody via a peptide linker. The human IgG1 knob-into-hole heavy chain pair also contains L234A, L235A, G237A mutations to abolish binding to FcγR and C1q, but retain FcRn binding for pharmacokinetics (PK). The structure of the PD1 Ab-IL-2 immunocytokine is depicted in Figure 3B, and exemplary immunocytokines are listed in Table 24. TIFF2025531804000027.tif64170

[0282] As will be appreciated by those skilled in the art, any optimized PD1 antibody disclosed in the present invention, including those having the sequences set forth in SEQ ID NOS: 44-49, can be used to construct PD1 Ab-IL-2 immunocytokines within the spirit and scope of the present invention. Similarly, any IL-2 variants with varying degrees of reduced potency disclosed in the present invention, particularly those having the sequences set forth in SEQ ID NOS: 151-180, can serve as building blocks for constructing PD1-targeted IL-2 immunocytokines. These designs aim to potentiate and / or augment PD1 antibody-based therapies for a wide range of cancers.

[0283] All genes were codon-optimized for expression in mammalian cells, synthesized, and subsequently subcloned into recipient mammalian expression vectors through the services of GenScript. Constructs were produced by co-transfecting the expression vectors into ExpiCHO cells (ThermoFisher) according to the manufacturer's instructions. Protein purification and characterization were performed according to the same procedures detailed in Example 2.

[0284] As expected, the potency of these IL-2 variants originally observed in the Fc fusion format was faithfully preserved when combined with PD1 antibodies, as demonstrated in a human PBMC assay measuring Ki67 expression. Moreover, these exemplary immunocytokines listed in Table 24 maintained the binding and PD1 blocking activity of their component PD1 antibody, P-1271 (SEQ ID NOs: 49 and 44), as confirmed in a Promega PD1 / PD-L1 blocking reporter assay.

[0285] Mouse PD1 Ab-IL-2 immunocytokines were similarly prepared for in vivo tumor models in immunocompetent mice. The IL-2 variant was fused to the C-terminus of the anti-mouse PD1 HC chain 2 (SEQ ID NO: 190) of the heterodimeric heavy chain pair (SEQ ID NOs: 189 and 190) via a GS linker (SEQ ID NO: 114). The light chain of the mouse PD1 antibody has the sequence set forth in SEQ ID NO: 52. Exemplary mouse PD1 Ab IL-2 immunocytokines, specifically P-0782, P-0786, and P-0783, feature IL-2 variants with P65R / C125I mutations (SEQ ID NO: 118), L19Q / P65R / C125I mutations (SEQ ID NO: 152), and L19H / P65R / C125I mutations (SEQ ID NO: 151), respectively. P-0837, containing the IL-2 domain of SEQ ID NO: 117, served as a wild-type IL-2 immunocytokine control.

[0286] P-0782, P-0786, and P-0783 were subsequently evaluated for activity in stimulating Ki67 expression in CD8+ T and NK cells using human PBMCs. The degree of potency reduction in P-0786 and P-0783 compared to P-0782 resulting from the incorporation of the L19 mutation (shown in Figures 31A and 31B) was similar to their respective Fc-fusion counterparts P-0759 and P-0731 compared to P-0704, as shown in Figures 18A and 18B.

[0287] The proliferative activity of P-0782, P-0786, and P-0783 was further evaluated using CTLL-2 cells, cytotoxic T cells derived from C57BL / 6 mice. Briefly, CTLL2 cells were harvested, washed, and resuspended in IL-2-free medium (RPMI1640, 10% FCS, 2 mM glutamine) for a 2-hour starvation period. After starvation, 50,000 cells / mL were transferred to 96-well U-bottom plates. Serial dilutions of PD1 Ab-IL-2 immunocytokine were then added, followed by 2 days of incubation. Cell proliferation was assessed using CellTiter-Glo (Promega) according to the manufacturer's instructions, and luminescence signals were measured. As depicted in Figure 31C, the reduced potency caused by the L19Q mutation in P-0786 and the L19H mutation in P-0783 compared to P-0782 was consistent between mouse-derived cells and human primary cells. This consistency highlights the mouse as a reliable model for analyzing the impact of varying IL-2 potency on in vivo pharmacodynamics and antitumor efficacy.

[0288] Example 19 Pharmacodynamic effects of PD1 Ab IL-2 immunocytokine in mice The pharmacodynamic effects of murine PD1 Ab-IL-2 immunocytokine were evaluated in C57BL / 6 mice using a single dose administration. Seven-week-old female C57BL / 6 mice from Charles River Laboratory were given a 7-day acclimation period before the start of the study. On day 0, mice received an intraperitoneal injection of either vehicle or one of the test compounds: P-0837, P-0782, P-0783, or P-0786. Blood samples were collected on days 0, 3, 5, 7, and 10 post-injection. Each group consisted of five mice. Immune profiling of heparinized whole blood was performed according to the procedure outlined in Example 14.

[0289] After a single injection at 2 mg / kg, striking differences in CD8 and NK cell expansion were observed among the test compounds. P-0782, which contains mutations that abolish IL-2Rα binding but do not affect IL-2Rβγ interactions, exhibited vigorous expansion of CD8+ T cells (Figure 32A) and NK cells (Figure 32B). Expansion of these lymphocyte subsets began on day 3 and peaked on day 7 with a dramatic 68-fold increase in CD8+ T cells and a 182-fold increase in NK cells. In sharp contrast, P-0837, which served as the wild-type control, exhibited a much more modest response. As shown in Figures 32A and 32B, peak cell expansion for both lymphocytes occurred on day 5 with a smaller increase of 3.9-fold for CD8+ T cells and 6.8-fold for NK cells.

[0290] Mutations that prevent IL-2Rα binding could potentially minimize the IL-2Rα (CD25) sink effect, subsequently increasing the availability of IL-2Rβγ. This enriched receptor engagement triggers vigorous expansion of cytotoxic cells, as shown with the IL-2Rβγ-selective full agonist P-0782. It is hypothesized that IL-2 mutations designed to reduce but not abolish IL-2Rα binding (as depicted in Figure 15B) could fine-tune regulatory T cell responses. The correct amount of modulation in IL-2Rα binding could establish an immune balance to improve systemic tolerability without compromising tumor-killing efficacy.

[0291] Figures 32A and 32B further demonstrate the pharmacodynamics of P-0783 and P-0786 after a 2 mg / kg dose. Compared to P-0782, the maximal responses observed with P-0786 and P-0783 were significantly reduced, consistent with an overall attenuation of efficacy. However, in contrast to P-0837, P-0786 exhibited a significantly prolonged and enhanced dose-response effect on cell expansion. The increases in both CD8+ T cell and NK cell numbers were delayed but sustained and durable. Peak responses were observed on day 7, demonstrating an 8.6-fold increase in CD8+ T cells and a 13-fold increase in NK cells. These numbers had not returned to baseline levels by day 10. P-0783, a weaker IL-2 agonist, showed a similar delayed but durable effect, leading to a 5.5-fold expansion in CD8+ T cells and a 14-fold expansion in NK cells in a dose-dependent manner (Figures 32A and 32B).

[0292] Additionally, Figure 32C highlights the direct correlation between efficacy levels, cytotoxic lymphocyte expansion, and resulting weight loss in mice. In particular, P-0782, an IL-2Rβγ-selective full agonist, induced dramatic increases in both CD8+ T cell and NK cell numbers, leading to the most substantial weight loss among the tested compounds. The attenuated agonists, P-0786 and P-0783, showed enhanced in vivo tolerability. Of these, P-0783 appeared slightly more tolerable than P-0786, consistent with its characterization as the weaker agonist of the two.

[0293] In summary, P-0782 demonstrated robust pharmacodynamic effects by significantly promoting the proliferation and expansion of CD8+ T and NK cells. P-0786 and P-0783 showed weaker effects, but their responses were durable. In vitro and in vivo efficacy assessments of these compounds were generally consistent. Notably, the attenuated potency of P-0786 and P-0783 translated into improved in vivo tolerability compared with the fully active P-0782. These findings support our design premise that attenuated cytokine potency helps mitigate pathway overactivation and alleviate antigen sink- and target-mediated deposition, ultimately reducing toxicity and improving pharmacokinetic and pharmacodynamic outcomes.

[0294] Example 20 In vivo efficacy of PD1 Ab IL-2 immunocytokine in a syngeneic mouse tumor model The antitumor efficacy of PD1 Ab-IL-2 immunocytokines, including P-0837, P-0782, P-0783, and P-0786, was investigated in the MC38 mouse colon cancer model. MC38 cells were implanted subcutaneously into 7- to 9-week-old female C57BL / 6 mice. Approximately 2 weeks later, tumors grew to approximately 75 mm. 3 Once tumors reached a mean volume of 1500 mm, mice were randomized (n=8) and treated with 0.5 mg / kg immunocytokine every 12 days (Q12D) for two injections. Vehicle (PBS) was included as a control. Tumor size and mouse weight were monitored twice weekly. When tumors reached a mean volume of 1500 mm, mice were randomized (n=8) and treated with 0.5 mg / kg immunocytokine every 12 days (Q12D) for two injections. Vehicle (PBS) was included as a control. Tumor size and mouse weight were monitored twice weekly. 3 Animals were euthanized when 0.1 mg / kg / day was reached or exceeded or when necrosis occurred.

[0295] In Figure 33A, the mean tumor volume as a function of time is presented for each group along with the standard error of the mean (SEM). The vehicle group (PBS-treated) showed rapid tumor growth. P-0837, a PD1 Ab immunocytokine with wild-type IL-2 equivalent, only inhibited tumor growth by 37% compared to the vehicle group. However, the other PD1 Ab-IL-2 immunocytokine, P-0782, P-0783, and P-0786, all significantly reduced tumor growth by 85–90% by day 26 posttreatment. Further analysis of individual tumor volumes (represented by each dot) at day 26 (Figure 33B) revealed that despite similar tumor growth inhibition, P-0782 only rendered two mice tumor-free, whereas P-0783 and P-0786 each resulted in five tumor-free cases. Both of the latter two have attenuated IL-2 efficacy. It is hypothesized that the full IL-2 agonist P-0782 causes pathway overactivation, target-mediated deposition, activation-induced cell death, and upregulation of inhibitory signals in T cells, ultimately leading to reduced antitumor efficacy in vivo. Therefore, only PD1 Ab-IL-2 immunocytokines with attenuated IL-2 will be further investigated for in vivo antitumor efficacy.

[0296] In a similarly performed experiment shown in Figure 34, both P-0783 and P-0786 showed significant tumor growth inhibition at a low dosage of 0.3 mg / kg (two doses at Q10D). At day 41, 30 days after the second and final treatment, five of eight mice treated with P-0783 and six of eight mice treated with P-0786 were tumor-free. At this dosage, P-0786 exhibited slightly better antitumor efficacy compared to P-0783.

[0297] Figures 35A and 35B show the progression of tumor volume and body weight change over time in mice treated with two Q10D doses of either P-0782 or the murine PD1 antibody P-0722. At a dosage of 9 mg / kg, P-0722 showed minimal efficacy. In sharp contrast, a significant and prolonged antitumor response was observed even at a nine-fold reduced dosage (1 mg / kg) of P-0786. By day 45, 34 days after the final treatment, all mice treated with P-0786 showed no tumor growth. Furthermore, no significant weight loss was observed with 1 mg / g P-0786 (Figure 35B). These findings highlight the pivotal role of the IL-2 component in enhancing the antitumor efficacy of PD1 Ab-IL-2 immunocytokine.

[0298] Furthermore, the dose response of P-0786 in inhibiting MC38 tumor growth was evaluated. Mice bearing established MC38 tumors (approximately 75 mm 3 The mean tumor volumes of mice (n=8) were measured by intraperitoneal administration of two doses of P-0786 at 0.03, 0.1, 0.3, and 1 mg / kg. Figure 36A shows the mean tumor volume ± SEM for each group over time, and Figures 36B-36E show the individual tumor growth curves for each dose group. The mean tumor size ± SEM for the vehicle group (represented by the dotted line) is shown for comparison. Both the 0.03 and 0.1 mg / kg doses resulted in a small TGI of 36%, and no mice exhibited complete tumor regression by day 21. However, a significant dose response emerged when the dose was increased from 0.1 mg / kg to 1 mg / kg in approximately three-fold increments. At 0.3 mg / kg, an 81% TGI was achieved by day 21, and two of the eight mice were tumor-free. Impressively, at the 1 mg / kg dosage, all 8 mice showed complete tumor eradication.

[0299] Ten mice that did not show tumor growth (two from the 0.3 mg / kg dose and eight from the 1 mg / kg dose) were rechallenged with MC38 cells 109 days after the initial implantation or 94 days after the first P-0786 dose. Figure 37 reveals that none of these rechallenged mice had tumor recurrence, unlike age-matched naive mice used as controls that successfully developed tumors. These findings suggest that the PD1 Ab IL-2 immunocytokine successfully induced long-term immunity.

[0300] The efficacy of P-0786 was also evaluated in two other syngeneic tumor models: the murine CT26 colon carcinoma and the B16F10 murine melanoma model. In the CT26 model, 5x10 5 The B16F10 model was performed by implanting 5 x 10 CT26 cells into female C57BL / 6 mice. 5 Tumors were similarly established by implanting 1000 B16F10 cells. CT26 tumor-bearing mice received two doses of P-0786 (0.6 mg / kg and 2 mg / kg) every 12 days, while B16F10 tumor-bearing mice received the same dosage every 10 days. All mice were monitored regularly with tumor measurements twice weekly.

[0301] Figure 38 demonstrates the dose-dependent, single-agent antitumor efficacy of P-0786 in both the CT26 (Figure 38A) and B16F10 (Figure 38B) models. In the CT26 model, there was strong tumor growth inhibition at both doses (65% TGI at 0.6 mg / kg and 91% TGI at 2 mg / kg by day 21). By day 41 after the first treatment, one of seven mice in the 0.6 mg / kg group and two of seven mice in the 2 mg / kg group were tumor-free. The B16F10 model, which grows vigorously and is less responsive to PD1 treatment than the MC38 model, showed initial tumor growth delay with P-0786 (35% TGI at 0.6 mg / kg and 58% TGI at 2 mg / kg), but tumors eventually developed in all mice (Figure 38B).

[0302] In summary, PD1 Ab-IL-2 immunocytokines effectively suppressed tumor growth across multiple syngeneic mouse tumor models. Compared with IL-2Rβγ-selective full agonists, attenuated IL-2, achieved by disrupting IL-2Rβ interactions, demonstrated improved in vivo tolerability and enhanced single-agent antitumor efficacy. PD1 Ab-IL-2 immunocytokines featuring IL-2 mutations that interfere with γc interactions are expected to have similar improvements. Given the distinct expression profile of γc in peripheral cells, IL-2 variants with attenuated γc activity may confer the additional benefits of reduced target sink and enhanced bioavailability.

[0303] All of the articles and methods disclosed and claimed in this application can be made and executed without undue experimentation in light of the present disclosure. While the articles and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the articles and methods without departing from the spirit and scope of the invention. All such variations and equivalents apparent to those skilled in the art, whether now existing or later developed, are deemed to be within the spirit and scope of the invention as defined by the appended claims. All patents, patent applications, and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All patents, patent applications, and publications are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for any and all purposes. The invention illustratively described herein may suitably be practiced in the absence of any element not specifically disclosed herein. Therefore, while the present invention has been particularly disclosed by preferred embodiments and optional features, it is to be understood that modifications and variations of the concepts disclosed herein may be undertaken by those skilled in the art, and that such modifications and variations are considered to be within the scope of the present invention as defined by the appended claims.

[0304] Sequence Listing The amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and one-letter codes for amino acids, as defined in 37 CFR 1.822. SEQ ID NO: 1 is the amino acid sequence of the mature human PD1 polypeptide. SEQ ID NOs: 2 to 5 are the amino acid sequences of the light chain variable domains of human PD1-blocking antibodies. SEQ ID NOs: 6 to 18 are the amino acid sequences of the heavy chain variable domains of human PD1-blocking antibodies. SEQ ID NOs: 19 to 21 are the amino acid sequences of the light chain CDR1 of a human PD1 blocking antibody. SEQ ID NOs: 22 to 24 are the amino acid sequences of the light chain CDR2 of a human PD1 blocking antibody. SEQ ID NO: 25 is the amino acid sequence of the human PD1 blocking antibody light chain CDR3. SEQ ID NO: 26 is the amino acid sequence of human PD1 blocking antibody heavy chain CDR1. SEQ ID NOs: 27 to 32 are the amino acid sequences of the heavy chain CDR2 of a human PD1 blocking antibody. SEQ ID NO: 33 is the amino acid sequence of the human PD1 blocking antibody heavy chain CDR3. SEQ ID NO: 34 is the amino acid sequence of the human kappa light chain constant domain. SEQ ID NO: 35 is the amino acid sequence of a human IgG1 heavy chain constant domain containing the L234A / L235A / G237A mutations. SEQ ID NO: 36 is the amino acid sequence of the human IgG4 heavy chain constant domain containing the S228P mutation. SEQ ID NO: 37 is the amino acid sequence of human immunoglobulin germline exon HGHV1-2 (GenBank accession number: X62106). SEQ ID NO: 38 is the amino acid sequence of human immunoglobulin germline exon HGHV3-23 (GenBank Accession No.: M99660). SEQ ID NO: 39 is the amino acid sequence of human immunoglobulin germline exon HGKV3D-11 (GenBank accession number: X17264). SEQ ID NO: 40 is the amino acid sequence of the human antibody heavy chain variable domain having GenBank Accession No.: AB063829. SEQ ID NO: 41 is the amino acid sequence of the human antibody light chain variable domain having GenBank accession number: M29469. SEQ ID NO: 42 is the amino acid sequence of the light chain of the reference human PD1 blocking antibody P-0734. SEQ ID NO: 43 is the amino acid sequence of the heavy chain of the reference human PD1 blocking antibody P-0734. SEQ ID NO: 44 is the amino acid sequence of the light chain of a human PD1 blocking antibody. SEQ ID NO: 45 is the amino acid sequence of the heavy chain of the human PD1 blocking antibody P-1174. SEQ ID NO: 46 is the amino acid sequence of the heavy chain of the human PD1 blocking antibody P-1194. SEQ ID NO: 47 is the amino acid sequence of the heavy chain of the human PD1 blocking antibody P-1201. SEQ ID NO: 48 is the amino acid sequence of the heavy chain of the human PD1 blocking antibody P-1238. SEQ ID NO: 49 is the amino acid sequence of the heavy chain of the PD1 human blocking antibody P-1271. SEQ ID NO: 50 is the amino acid sequence of the light chain of the benchmark human PD1 blocking antibody P-0795. SEQ ID NO: 51 is the amino acid sequence of the heavy chain of the benchmark human PD1 blocking antibody P-0795. SEQ ID NO: 52 is the amino acid sequence of the light chain of the surrogate murine PD1 blocking antibody P-0722. SEQ ID NO: 53 is the amino acid sequence of the heavy chain of the surrogate murine PD1 blocking antibody P-0722. SEQ ID NOs: 54 to 77 are the amino acid sequences of various protease substrate peptides. SEQ ID NOs: 78-94 are the amino acid sequences of various protease-cleavable linkers comprising various spacer peptides flanking a protease substrate peptide. SEQ ID NOs: 95-115 are the amino acid sequences of various non-cleavable linker sequences. SEQ ID NO: 116 is the amino acid sequence of the mature form of human IL-2. SEQ ID NOs: 117 to 180 are the amino acid sequences of human IL-2 variant polypeptides. SEQ ID NO: 181 is the human IL-2Rα amino acid sequence. SEQ ID NO: 182 is the human IL-2Rα sushi domain amino acid sequence. SEQ ID NOs: 183 to 185 are the amino acid sequences of human IL-2Rα sushi domain variant polypeptides. SEQ ID NO: 186 is the amino acid sequence of human IgG1 Fc containing the L234A / L235A / G237A mutations. SEQ ID NO: 187 is the amino acid sequence of human IgG1 knob-Fc containing the L234A / L235A / G237A mutations. SEQ ID NO: 188 is the amino acid sequence of human IgG1 whole-Fc containing the L234A / L235A / G237A mutations. SEQ ID NOs: 189 and 190 are the amino acid sequences of the heterodimeric heavy chains of surrogate murine PD1 Ab P-0722. SEQ ID NOs: 191 and 192 are the amino acid sequences of the heterodimeric heavy chains of germline antibody P-1260. SEQ ID NO: 193 is the amino acid sequence of the light chain of germline antibody P-1260. SEQ ID NOs: 194-209 are the amino acid sequences of the heavy chains of various human PD1 Abs and / or human PD1 Ab-IL-2 VitoKines. SEQ ID NOs: 210-215 are the amino acid sequences of the heavy chains of various human PD1 Ab-IL-2 immunocytokines. Sequence Listing Human PD1 mature protein sequence FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 1) Sequence of the human PD1-blocking antibody light chain variable domain EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR (SEQ ID NO: 2) Sequence of the human PD1-blocking antibody light chain variable domain EIVLTQSPATLSLSPGERATLSCRASQGVSTSGYSYLHWYQQKPGQAPRLLIYLASYRESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR (SEQ ID NO: 3) Sequence of the human PD1-blocking antibody light chain variable domain EIVLTQSPATLSLSPGERATLSCRASQGVSTSGYSYLHWYQQKPGQAPRLLIYLASYRASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR (SEQ ID NO: 4) Sequence of the human PD1-blocking antibody light chain variable domain EIVLTQSPATLSLSPGERATLSCRASQGVSTSGYSYLAWYQQKPGQAPRLLIYLASYRASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR (SEQ ID NO: 5) Sequence of the human PD1-blocking antibody heavy chain variable domain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 6) Sequence of the human PD1-blocking antibody heavy chain variable domain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFAQKFQGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 7) Sequence of the human PD1-blocking antibody heavy chain variable domain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNYAQKFQGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 8) Sequence of the human PD1-blocking antibody heavy chain variable domain QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFAQKFQGRVTLTTDSSTSTAYMELSSLRSDDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 9) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNYADKFKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 10) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNYADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 11) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWMGGINPSNGGTNYADKFKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 12) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWMGGINPSNGGTNYADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 13) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 14) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 15) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFKGRFTISRDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 16) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFKGRFTISTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 17) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 18) Human PD1-blocking antibody CDR-L1 sequence RASKGVSTSGYSYLH (SEQ ID NO: 19) Human PD1-blocking antibody CDR-L1 sequence RASQGVSTSGYSYLH (SEQ ID NO: 20) Human PD1-blocking antibody CDR-L1 sequence RASQGVSTSGYSYLA (SEQ ID NO: 21) Sequence of human PD1-blocking antibody CDR-L2 YLASYLES (SEQ ID NO: 22) Sequence of human PD1-blocking antibody CDR-L2 YLASYRES (SEQ ID NO: 23) Sequence of human PD1-blocking antibody CDR-L2 YLASYRAS (SEQ ID NO: 24) Sequence of human PD1-blocking antibody CDR-L3 QHSRDLPLT (SEQ ID NO: 25) Human PD1-blocking antibody CDR-H1 sequence NYYMY (SEQ ID NO: 26) Human PD1 blocking antibody CDR-H2 sequence GINPSNGGTNFNEKFKN (SEQ ID NO: 27) Human PD1 blocking antibody CDR-H2 sequence GINPSNGGTNFAQKFQG (SEQ ID NO: 28) Human PD1 blocking antibody CDR-H2 sequence GINPSNGGTNYAQKFQG (SEQ ID NO: 29) Human PD1 blocking antibody CDR-H2 sequence GINPSNGGTNYADKFKG (SEQ ID NO: 30) Human PD1 blocking antibody CDR-H2 sequence GINPSNGGTNFADKFKG (SEQ ID NO: 31) Human PD1 blocking antibody CDR-H2 sequence GINPSNGGTNFNDSVKG (SEQ ID NO: 32) Human PD1 blocking antibody CDR-H3 sequence RDYRFDMGFDY (SEQ ID NO: 33) Human kappa light chain constant domain sequence TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 34) Sequence of the human IgG1 constant domain with the L234A / L235A / G237A mutations ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 35) Sequence of the human IgG4 constant domain with the S228P mutation ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 36) Human antibody germline IGHV1-2 sequences QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR (SEQ ID NO: 37) Human antibody germline IGHV3-23 sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO: 38) Human antibody germline IGKV3D-11 sequence EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSNWH (SEQ ID NO: 39) Human antibody GenBank number: AB063829 sequence QVQLVQSGVEVKKPGASVKVSCKASGYTFTSNAISWVRQAPGQGLEWMGWISTYKGKANYAQKFQDRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARWRAVVGRGGGLDVWGQGTTVTVSS (SEQ ID NO: 40) Human antibody GenBank number: M29469 sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNKATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSKWPLTFGGGTKVEIKG (SEQ ID NO: 41) Reference antibody P-0734 light chain sequence EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 42) Reference antibody P-0734 heavy chain sequence QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 43) Sequence of the human PD1-blocking Ab light chain EIVLTQSPATLSLSPGERATLSCRASQGVSTSGYSYLHWYQQKPGQAPRLLIYLASYRESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 44) Sequence of the heavy chain of human PD1-blocking Ab P-1174 QVQLVQSVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFAQKFQGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 45) Sequence of the heavy chain of the human PD1-blocking antibody P-1194 EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNYADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 46) Sequence of the heavy chain of the human PD1-blocking antibody P-1201 EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 47) Sequence of the heavy chain of the human PD1-blocking antibody P-1238 EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWMGGINPSNGGTNYADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 48) Sequence of the heavy chain of human PD1-blocking Ab P-1271 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFAQKFQGRVTLTTDSSTSTAYMELSSLRSDDTAVYYCARRDYRFDMGFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 49) Light chain sequence of the benchmark human PD1 blocking antibody P-0795 DIVMTQSPLSLPVTPGEPASITCKASQDVETVVAWYLQKPGQSPRLLIYWASTRHTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYSRYPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 50) Sequence of the heavy chain of the benchmark human PD1 blocking antibody P-0795 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATISGGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPDSSSGVAYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 51) Sequence of the light chain of the surrogate mouse PD1 blocking antibody P-0722 DIVMTQGTLPNPVPSGESVSITCRSSKSLLYSDGKTYLNWYLQRPGQSPQLLIYWMSTRASGVSDRFSGSGSGTDFTLKISGVEAEDVGIYYCQQGLEFPTFGGGTKLELKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPRDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 52) Sequence of the heavy chain of the surrogate mouse PD1 blocking antibody P-0722 EVQLQESGPGLVKPSQSLSLTCSVTGYSITSSYRWNWIRKFPGNRLEWMGYINSAGISNYNPSLKRRISITRDTSKNQFFLQVNSVTTEDAATYYCARSDNMGTTPFTYWGQG TLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKP CICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISKTKGGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG (SEQ ID NO: 53) Protease substrate peptide sequence SPLGLAGS (SEQ ID NO: 54) Protease substrate peptide sequence EPLELRAG (SEQ ID NO: 55) Protease substrate peptide sequence LSGRSDNH (SEQ ID NO: 56) Protease substrate peptide sequence GPLGIAGQ (SEQ ID NO: 57) Protease substrate peptide sequence GTAHLMGG (SEQ ID NO: 58) Protease substrate peptide sequence RIGSLRTA (SEQ ID NO: 59) Protease substrate peptide sequence SGRSENIRTA (SEQ ID NO: 60) Protease substrate peptide sequence GPLGMLSQ (SEQ ID NO: 61) Protease substrate peptide sequence GPAGMKGL (SEQ ID NO: 62) Protease substrate peptide sequence RPSASRSA (SEQ ID NO: 63) Protease substrate peptide sequence PLGLAG (SEQ ID NO: 64) Protease substrate peptide sequence LGGSGRSANAILE (SEQ ID NO: 65) Protease substrate peptide sequence GGSGRSANAI (SEQ ID NO: 66) Protease substrate peptide sequence SGRSA (SEQ ID NO: 67) Protease substrate peptide sequence AANL (SEQ ID NO: 68) Protease substrate peptide sequence GPTNKVR (SEQ ID NO: 69) Protease substrate peptide sequence GFFY (SEQ ID NO: 70) Protease substrate peptide sequence GPICFRLG (SEQ ID NO: 71) Protease substrate peptide sequence RQAGFSL (SEQ ID NO: 72) Protease substrate peptide sequence RQARAVGG (SEQ ID NO: 73) Protease substrate peptide sequence PMAKK (SEQ ID NO: 74) Protease substrate peptide sequence HSSKLQ (SEQ ID NO: 75) Protease substrate peptide sequence GPLGMLSQPMAKK (SEQ ID NO: 76) Protease substrate peptide sequence PMAKKGPLGMLSQ (SEQ ID NO: 77) Protease-cleavable linker sequence GGGSGGGGSGGGGSLSGRSDNHGGSGGGGS (SEQ ID NO: 78) Protease-cleavable linker sequence GSSSGRSENIRTAGT (SEQ ID NO: 79) Protease-cleavable linker sequence GGGGSGGGGSGGGSLGGSGRSANAILEGGSGGGGS (SEQ ID NO: 80) Protease-cleavable linker sequence GGGGSGGGGSLGGSGRSANAILEGGGGS (SEQ ID NO: 81) Protease-cleavable linker sequence GGGGSLGGSGRSANAILEGGS (SEQ ID NO: 82) Protease-cleavable linker sequence GGGSGPTNKVRGGS (SEQ ID NO: 83) Protease-cleavable linker sequence GGSGPLGMLSQGGGS (SEQ ID NO: 84) Protease-cleavable linker sequence GGPLGMLSQS (SEQ ID NO: 85) Protease-cleavable linker sequence GGGPLGMLSQGGS (SEQ ID NO: 86) Protease-cleavable linker sequence GGPTNKVRGS (SEQ ID NO: 87) Protease-cleavable linker sequence GRQARAVGGS (SEQ ID NO: 88) Protease-cleavable linker sequence GGGSGRSENIRTAGG (SEQ ID NO: 89) Protease-cleavable linker sequence SGGPGPAGMKGLPGS (SEQ ID NO: 90) Protease-cleavable linker sequence GGGGSPMAKKGGGGS (SEQ ID NO: 91) Protease-cleavable linker sequence GGPLGMLSQPMAKKS (SEQ ID NO: 92) Protease-cleavable linker sequence GGSGPLGMLSQPMAKKGGGS (SEQ ID NO: 93) Protease-cleavable linker sequence GGGPMAKKGPLGMLSQGGGS (SEQ ID NO: 94) Non-cleavable linker sequence EPKSSDKTHTSPPS (SEQ ID NO: 95) Non-cleavable linker sequence GGGSGGGSGGGS (SEQ ID NO: 96) Non-cleavable linker sequence GGGS (SEQ ID NO: 97) Non-cleavable linker sequence GSSGGSGGS (SEQ ID NO: 98) Non-cleavable linker sequence GSSGT (SEQ ID NO: 99) Non-cleavable linker sequence GGGGSGGGGSGGGS (SEQ ID NO: 100) Non-cleavable linker sequence AEAAAKEAAAKEAAAKA (SEQ ID NO: 101) Non-cleavable linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 102) Non-cleavable linker sequence GGGSGGGS (SEQ ID NO: 103) Non-cleavable linker sequence GSGG (SEQ ID NO: 104) Non-cleavable linker sequence GGSS (SEQ ID NO: 105) Non-cleavable linker sequence GGGGS (SEQ ID NO: 106) Non-cleavable linker sequence GGSGG (SEQ ID NO: 107) Non-cleavable linker sequence SGGG (SEQ ID NO: 108) Non-cleavable linker sequence GSGS (SEQ ID NO: 109) Non-cleavable linker sequence GSGSGS (SEQ ID NO: 110) Non-cleavable linker sequence GSGSGSGS (SEQ ID NO: 111) Non-cleavable linker sequence GSGSGSGSGS (SEQ ID NO: 112) Non-cleavable linker sequence GSGSGSGSGSGS (SEQ ID NO: 113) Non-cleavable linker sequence GGGGSGGGGS (SEQ ID NO: 114) Non-cleavable linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 115) Sequence of the naturally occurring mature form of human IL-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 116) Sequence of human IL-2 C125I variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 117) Sequence of the human IL-2 P65R / C125I variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 118) Sequence of human IL-2 P65K / C125I variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKKLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 119) Sequence of human IL-2 P65N / C125I variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKNLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 120) Sequence of the human IL-2 P65Q / C125I variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKQLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 121) Sequence of the human IL-2 P65H / C125I variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKHLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 122) Sequence of the human IL-2 P65G / C125I variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKGLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 123) Sequence of the human IL-2 P65E / C125I variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKELEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 124) Sequence of human IL-2 P65A / C125I variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKALEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 125) Sequence of human IL-2 L19H / C125I variant APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 126) Sequence of human IL-2 L19Q / C125I variant APTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 127) Sequence of human IL-2 L19Y / C125I variant APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 128) Human IL-2 L19D / C125I variant sequence APTSSSTKKTQLQLEHLLDDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 129) Sequence of human IL-2 L19S / C125I variant APTSSSTKKTQLQLEHLLSDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 130) Sequence of human IL-2 L19N / C125I variant APTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 131) Sequence of human IL-2 L19R / C125I variant APTSSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 132) Sequence of human IL-2 C125I / Q126A variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIASIISTLT (SEQ ID NO: 133) Sequence of human IL-2 C125I / Q126D variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIDSIISTLT (SEQ ID NO: 134) Sequence of human IL-2 C125I / Q126E variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT (SEQ ID NO: 135) Sequence of human IL-2 C125I / Q126F variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIFSIISTLT (SEQ ID NO: 136) Sequence of human IL-2 C125I / Q126G variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIGSIISTLT (SEQ ID NO: 137) Sequence of human IL-2 C125I / Q126H variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIHSIISTLT (SEQ ID NO: 138) Sequence of human IL-2 C125I / Q126I variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIISIISTLT (SEQ ID NO: 139) Sequence of human IL-2 C125I / Q126K variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 140) Sequence of human IL-2 C125I / Q126L variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFILSIISTLT (SEQ ID NO: 141) Sequence of human IL-2 C125I / Q126M variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIMSIISTLT (SEQ ID NO: 142) Sequence of human IL-2 C125I / Q126N variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFINSIISTLT (SEQ ID NO: 143) Sequence of human IL-2 C125I / Q126P variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIPSIISTLT (SEQ ID NO: 144) Sequence of human IL-2 C125I / Q126R variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIRSIISTLT (SEQ ID NO: 145) Sequence of human IL-2 C125I / Q126S variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFISSIISTLT (SEQ ID NO: 146) Sequence of human IL-2 C125I / Q126T variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFITSIISTLT (SEQ ID NO: 147) Sequence of human IL-2 C125I / Q126V variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIVSIISTLT (SEQ ID NO: 148) Sequence of human IL-2 C125I / Q126W variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIWSIISTLT (SEQ ID NO: 149) Sequence of human IL-2 C125I / Q126Y variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIYSIISTLT (SEQ ID NO: 150) Sequence of human IL-2 L19H / P65R / C125I variant APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 151) Sequence of human IL-2 L19Q / P65R / C125I variant APTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 152) Sequence of human IL-2 L19Y / P65R / C125I variant APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 153) Sequence of the human IL-2 L19Q / P65Q / C125I variant APTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKQLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 154) Sequence of the human IL-2 P65R / C125I / Q126A variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIASIISTLT (SEQ ID NO: 155) Sequence of the human IL-2 P65R / C125I / Q126D variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIDSIISTLT (SEQ ID NO: 156) Sequence of the human IL-2 P65R / C125I / Q126E variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKR LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT (SEQ ID NO: 157) Sequence of the human IL-2 P65R / C125I / Q126F variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKR LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIFSIISTLT (SEQ ID NO: 158) Sequence of the human IL-2 P65R / C125I / Q126G variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIGSIISTLT (SEQ ID NO: 159) Sequence of the human IL-2 P65R / C125I / Q126H variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIHSIISTLT (SEQ ID NO: 160) Sequence of the human IL-2 P65R / C125I / Q126I variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIISIISTLT (SEQ ID NO: 161) Sequence of the human IL-2 P65R / C125I / Q126K variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 162) Sequence of the human IL-2 P65R / C125I / Q126L variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFILSIISTLT (SEQ ID NO: 163) Sequence of the human IL-2 P65R / C125I / Q126M variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIMSIISTLT (SEQ ID NO: 164) Sequence of the human IL-2 P65R / C125I / Q126N variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFINSIISTLT (SEQ ID NO: 165) Sequence of the human IL-2 P65R / C125I / Q126P variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIPSIISTLT (SEQ ID NO: 166) Sequence of the human IL-2 P65R / C125I / Q126R variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIRSIISTLT (SEQ ID NO: 167) Sequence of the human IL-2 P65R / C125I / Q126S variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFISSIISTLT (SEQ ID NO: 168) Sequence of the human IL-2 P65R / C125I / Q126T variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFITSIISTLT (SEQ ID NO: 169) Sequence of the human IL-2 P65R / C125I / Q126V variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIVSIISTLT (SEQ ID NO: 170) Sequence of the human IL-2 P65R / C125I / Q126W variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIWSIISTLT (SEQ ID NO: 171) Sequence of the human IL-2 P65R / C125I / Q126Y variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIYSIISTLT (SEQ ID NO: 172) Sequence of the human IL-2 L19Y / P65R / C125I / Q126N variant APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFINSIISTLT (SEQ ID NO: 173) Sequence of human IL-2 L19Q / P65R / C125I / Q126H variant APTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIHSIISTLT (SEQ ID NO: 174) Sequence of human IL-2 L19Y / P65Q / C125I / Q126S variant APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKQLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFISSIISTLT (SEQ ID NO: 175) Sequence of the human IL-2 P65Q / C125I / Q126N variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKQLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFINSIISTLT (SEQ ID NO: 176) Sequence of the human IL-2 P65Q / C125I / Q126H variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKQLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIHSIISTLT (SEQ ID NO: 177) Sequence of the human IL-2 P65Q / C125I / Q126M variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKQLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIMSIISTLT (SEQ ID NO: 178) Sequence of the human IL-2 P65Q / C125I / Q126F variant APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQC...

Claims

1. 1. A bioactivatable polypeptide drug construct comprising, in N-terminal to C-terminal direction (D1-D2-D3): 1) a tumor infiltrating lymphocyte (TIL) targeting moiety D1 domain ("D1"), 2) a bioactivatable moiety D2 domain ("D2"), and 3) a concealment moiety D3 domain ("D3"); D1 functions to target said bioactivatable moiety to a site of intended treatment; D3 has the ability to conceal the functional activity of D2 until it is activated at said site of intended treatment; and D1 is an optimized PD1 blocking antibody, D2 is an IL-2 variant polypeptide, and D3 is an IL-2Rα sushi variant.

2. 1. A bioactivatable polypeptide drug construct comprising, in an N-terminal to C-terminal direction (D3-D2-D1), 1) a hiding moiety D3 domain ("D3"), 2) a bioactivatable moiety D2 domain ("D2"), and 3) a tumor infiltrating lymphocyte (TIL) targeting moiety D1 domain ("D1"), wherein D1 functions to target said bioactivatable moiety to a site of intended treatment; D3 has the ability to hide the functional activity of D2 until it is activated at said site of intended treatment; and D1 is an optimized PD1 blocking antibody, D2 is an IL-2 variant polypeptide, and D3 is an IL-2Rα sushi variant.

3. 3. The bioactivatable polypeptide drug construct of claim 1, wherein the optimized PD1-blocking antibody is selected from an antibody comprising: (a) a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 7; or (b) a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 9; or (c) a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 11; (d) a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 13; or (e) a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:

18.

4. 4. A bioactivatable polypeptide drug construct according to any one of claims 1 to 3, wherein domain D2 is an IL-2 variant polypeptide selected from the group of polypeptides having the amino acid sequences set forth in SEQ ID NOs: 117 to 180.

5. 5. The bioactivatable polypeptide drug construct of any one of claims 1 to 4, wherein domain D3 is an IL-2Rα sushi variant polypeptide selected from the group of polypeptides having the amino acid sequence set forth in SEQ ID NOs: 183 to 185.

6. 6. The construct of any one of claims 1 to 5, wherein the D1, D2 and D3 domains of the construct are each in the form of a monomer, each in the form of a dimer, or collectively in the form of a combination of a dimer and a monomer.

7. 7. The construct of any one of claims 1 to 6, wherein D2 is attached to D1 by a peptide linker ("L1") selected from the group consisting of a protease-cleavable peptide linker and a non-cleavable peptide linker.

8. The construct of claim 7, wherein the protease-cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 54-77 and 78-94.

9. The construct of claim 7, wherein the non-cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 95 to 115.

10. 10. The construct of any one of claims 1 to 9, wherein D2 is attached to D3 by a peptide linker ("L2") selected from the group consisting of a protease-cleavable peptide linker and a non-cleavable peptide linker.

11. The construct of claim 10, wherein the protease-cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 54-77 and 78-94.

12. The construct of claim 10, wherein the non-cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 95 to 115.

13. The construct of any one of claims 1 to 12, wherein L1 and L2 are both protease-cleavable peptide linkers.

14. The construct of any one of claims 1 to 12, wherein L1 and L2 are both non-cleavable peptide linkers.

15. The construct of any one of claims 1 to 12, wherein L1 is a protease-cleavable peptide linker and L2 is a non-cleavable peptide linker.

16. The construct of any one of claims 1 to 12, wherein L1 is a non-cleavable peptide linker and L2 is a protease-cleavable peptide linker.

17. A pharmaceutical composition comprising the construct of any one of claims 1 to 16 in admixture with a pharmaceutically acceptable carrier.

18. 20. A method of treating cancer or cancer metastasis in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 17.

19. 19. The method of claim 18, wherein the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, or rhabdomyosarcoma or any cancer.

20. 20. The method of any one of claims 18-19, further comprising a second therapeutic agent or treatment capable of treating cancer or cancer metastasis in the subject.

21. 21. The method of claim 20, wherein the second treatment is selected from the group consisting of cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, stem cell transplantation, cell therapy including CAR-T, CAR-NK, iPS-derived CAR-T or iPS-derived CAR-NK, and a vaccine, such as Bacillus Calmette-Guerin (BCG).

22. 22. The method of claim 21, wherein the immunotherapy is selected from the group consisting of: treatment using a depleting antibody against a specific tumor antigen; treatment using an antibody-drug conjugate; treatment using an agonist, antagonist, or blocking antibody against a costimulatory or co-inhibitory molecule (immune checkpoint), such as CTLA-4, PD-L1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec-7, Siglec-8, Siglec-9, Siglec-15, and VISTA; treatment using a bispecific T-cell engaging antibody (BiTE®), such as blinatumomab; treatment involving administration of a biological response modifier, such as IL-12, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ.

23. A nucleic acid molecule encoding the construct of any one of claims 1 to 16.

24. 24. An expression vector comprising the nucleic acid molecule of claim 23.

25. A host cell comprising the expression vector of claim 24.

26. 27. A method for producing a bioactivatable polypeptide drug construct according to any one of claims 1 to 16, comprising culturing a host cell according to claim 25 under conditions promoting expression of said bioactivatable polypeptide drug construct and recovering said bioactivatable polypeptide drug construct protein.

27. 27. An isolated bioactivatable polypeptide drug construct protein produced by the method of claim 26.

28. An isolated interleukin-2 (IL-2) fusion protein complex, comprising an IL-2 polypeptide (or a variant thereof) linked to an optimized PD1 blocking antibody to form an IL-2-PD1 blocking antibody fusion protein, wherein the optimized PD1 blocking antibody comprises: (a) a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:3, and a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:7; or (b) a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:3, and a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:9; or (c) an amino acid having the sequence set forth in SEQ ID NO:

3. (d) a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:13; or (e) a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:18, and wherein the optimized PD1 blocking antibody targets the IL-2-PD1 blocking antibody fusion protein to tumor infiltrating lymphocytes (TILs).

29. 29. The IL-2-PD1 blocking antibody fusion protein of claim 28, wherein the IL-2 polypeptide is linked to the C-terminus of the PD1 blocking antibody.

30. 30. The IL-2-PD1 blocking antibody fusion protein of any one of claims 28-29, wherein the IL-2 variant polypeptide is selected from the group of polypeptides having the amino acid sequence set forth in SEQ ID NOs: 117-180.

31. 31. The IL-2-PD1 blocking antibody fusion protein of any one of claims 28 to 30, wherein the IL-2 polypeptide is covalently attached to the PD1 blocking antibody by a peptide linker.

32. 32. The IL-2-PD1 blocking antibody fusion protein of claim 31, wherein the peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 54-115.

33. A pharmaceutical composition comprising the fusion protein of any one of claims 28 to 32 in admixture with a pharmaceutically acceptable carrier.

34. 34. A method of treating cancer or cancer metastasis in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 33.

35. 35. The method of claim 34, wherein the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, or rhabdomyosarcoma or any cancer.

36. 36. The method of any one of claims 34-35, further comprising a second therapeutic agent or treatment capable of treating cancer or cancer metastasis in the subject.

37. 37. The method of claim 36, wherein the second treatment is selected from the group consisting of cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, stem cell transplantation, cell therapy including CAR-T, CAR-NK, iPS-derived CAR-T or iPS-derived CAR-NK, and a vaccine, such as Bacillus Calmette-Guerin (BCG).

38. 38. The method of claim 37, wherein the immunotherapy is selected from the group consisting of: treatment using a depleting antibody against a specific tumor antigen; treatment using an antibody-drug conjugate; treatment using an agonist, antagonist, or blocking antibody against a costimulatory or co-inhibitory molecule (immune checkpoint), such as CTLA-4, PD-L1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec-7, Siglec-8, Siglec-9, Siglec-15, and VISTA; treatment using a bispecific T-cell engaging antibody (BiTE®), such as blinatumomab; treatment involving administration of a biological response modifier, such as IL-12, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ.

39. A nucleic acid molecule encoding the fusion protein of any one of claims 28 to 32.

40. 40. An expression vector comprising the nucleic acid molecule of claim 39.

41. A host cell comprising the expression vector of claim 40.

42. 43. A method of producing the isolated fusion protein of any one of claims 28 to 32, comprising culturing the host cell of claim 41 under conditions that promote expression of the fusion protein and recovering the isolated fusion protein.

43. 43. An isolated fusion protein produced by the method of claim 42.

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